Methods of producing nor-opioid and nal-opioid benzylisoquinoline alkaloids

ABSTRACT

A method of demethylizing an opioid to a nor-opioid is provided. The method comprises contacting an opioid with at least one enzyme. Contacting the opioid with the at least one enzyme converts the opioid to a nor-opioid. A method of converting a nor-opioid to a nal-opioid is provided. The method comprises contacting a nor-opioid with at least one enzyme. Contacting the nor-opioid with the at least one enzyme converts the nor-opioid to a nal-opioid.

CROSS-REFERENCE

This application is a continuation application of Ser. No. 16/127,084,filed Sep. 10, 2018, now U.S. Pat. No. 10,738,335, which is acontinuation application of International Patent Application No.PCT/US2017/057237, filed Oct. 18, 2017, which application claims thebenefit of U.S. Provisional Application No. 62/409,837, filed Oct. 18,2016, and U.S. Provisional Application No. 62/473,215, filed Mar. 17,2017, which applications are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 7, 2020, isnamed 47840706302_SL.txt and is 515,092 bytes in size.

BACKGROUND OF THE INVENTION

Medicinal opioids are used for treating moderate to severe pain, but mayexhibit addictive properties. Due to the mechanism by which medicinalopioids relieve pain, these medications are among the most effectivepainkillers in modern medicine. Additionally, however, medicinal opioidsare also widely abused. In addressing the use of medicinal opioids,policy makers are tasked with balancing the under-treatment of pain,while mitigating the risk for opioid abuse. Pharmacotherapies haveproven effective in treating and preventing opioid addiction but thehigh cost of these therapeutics is a limiting factor in the scope andreach of treatment programs.

SUMMARY OF THE INVENTION

The present disclosure provides methods for demethylating a first opioidto a second opioid. The present disclosure further provides methods fordemethylating an opioid to a nor-opioid. Additionally, the presentdisclosure provides methods for altering an opioid to a nal-opioid.Further, the present disclosure provides engineered cells for producinga nor-opioid from an opioid present within the engineered cell. Thepresent disclosure also provides engineered cells for producing anal-opioid from a nor-opioid present within the engineered cell.

An aspect of the invention provides a method for demethylating a firstopioid to a second opioid. The method comprises contacting the firstopioid with at least one enzyme, wherein contacting the first opioidwith the at least one enzyme converts the first opioid to a secondopioid through loss of an O-linked methyl group, wherein the firstopioid is not selected from the group consisting of codeine andthebaine.

Another aspect of the invention provides a method of demethylating anopioid to a nor-opioid. The method comprises contacting the first opioidwith at least one enzyme, wherein contacting the first opioid with theat least one enzyme converts the first opioid to a second opioid throughloss of an O-linked methyl group. The method also comprises contactingthe second opioid with at least one enzyme, wherein contacting theopioid with the at least one enzyme converts the second opioid to anor-opioid through loss of an N-linked methyl group.

An additional aspect of the invention provides another method ofdemethylating an opioid to a nor-opioid. The method comprises contactingthe opioid with at least one enzyme, wherein contacting the opioid withthe at least one enzyme converts the opioid to a nor-opioid throughremoval of an N-linked methyl group from the opioid, wherein the opioidis not thebaine when the opioid contacts the at least one enzyme invitro.

A further aspect of the invention provides a method of altering anopioid to a nal-opioid. The method comprises contacting the opioid withat least a first enzyme, wherein contacting the opioid with the at leasta first enzyme converts the opioid to a nor-opioid through removal of anN-linked methyl group from the opioid. The method also comprisescontacting the nor-opioid with at least a second enzyme, whereincontacting the nor-opioid with the at least a second enzyme in thepresence of a cofactor converts the nor-opioid to a nal-opioid throughtransfer of a sidechain from the cofactor.

Another aspect of the invention provides another method of altering anopioid to a nal-opioid. The method comprises contacting the first opioidwith at least one enzyme, wherein contacting the first opioid with theat least one enzyme converts the first opioid to a second opioid throughloss of an O-linked methyl group. The method also comprises contactingthe second opioid with at least a second enzyme, wherein contacting theopioid with the at least a second enzyme converts the second opioid to anor-opioid through loss of an N-linked methyl group. Additionally, themethod comprises contacting the nor-opioid with at least a third enzyme,wherein contacting the nor-opioid with the at least a third enzyme inthe presence of a cofactor converts the nor-opioid to a nal-opioidthrough transfer of a sidechain from the cofactor.

An additional aspect of the invention provides an engineered cell thatproduces a nor-opioid from an opioid present within the engineered cell,the engineered cell comprising a heterologous coding sequence encodingan N-demethylase produced by the engineered cell, wherein theN-demethylase converts the opioid within the engineered cell to thenor-opioid and wherein the nor-opioid is produced within the engineeredcell.

A further aspect of the invention provides an engineered cell thatproduces a nal-opioid from a nor-opioid present within the engineeredcell, the engineered cell comprising a heterologous coding sequenceencoding an N-methyltransferase produced by the engineered cell, whereinthe N-methyltransferase converts the nor-opioid within the engineeredcell to the nal-opioid.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates examples of synthesis, recycling, and salvagepathways of tetrahydrobiopterin, in accordance with embodiments of theinvention.

FIG. 2 illustrates a biosynthetic scheme for conversion of glucose to4-HPA, dopamine, and 3,4-DHPA, in accordance with embodiments of theinvention.

FIG. 3 illustrates a schematic example of (R)-1-benzylisoquinolinealkaloid formation, in accordance with embodiments of the invention.

FIG. 4 illustrates an amino acid sequence of a CYP-COR enzyme (SEQ IDNO: 1), in accordance with embodiments of the invention.

FIG. 5 illustrates a biosynthetic scheme for conversion of L-tyrosine toreticuline via norcoclaurine, in accordance with embodiments of theinvention.

FIG. 6 illustrates a biosynthetic scheme for conversion of L-tyrosine toreticuline via norlaudanosoline, in accordance with embodiments of theinvention.

FIG. 7 illustrates a biosynthetic scheme for conversion of L-tyrosine tomorphinan alkaloids, in accordance with embodiments of the invention.

FIG. 8 illustrates a biosynthetic scheme for production ofsemi-synthetic opioids, in accordance with embodiments of the invention.

FIG. 9 illustrates tyrosine hydroxylase mutants that improve reticulineproduction from sugar in engineered yeast strains, in accordance withembodiments of the invention.

FIG. 10 illustrates coexpression of dihydrofolate reductase (DHFR) thatimproves L-DOPA production by tyrosine hydroxylase in engineered yeaststrains, in accordance with embodiments of the invention.

FIG. 11A illustrates the addition of antioxidants to culture media thatimproves L-DOPA production by tyrosine hydroxylase in engineered yeaststrains.

FIG. 11B illustrates the addition of antioxidants to culture media thatincrease BH₄ levels, in accordance with embodiments of the invention.

FIG. 12A illustrates a biosynthetic scheme for conversion of L-tyrosineto bisBIAs.

FIG. 12B illustrates yeast strains engineered to biosynthesize bisBIAs,in accordance with embodiments of the invention.

FIG. 13 illustrates a phylogenetic tree of cytochrome P450oxidase-codeinone reductase-like (CYP-COR) fusions, in accordance withembodiments of the invention.

FIG. 14 illustrates an LC/MS-MS analysis of yeast strains engineered toconvert (S)-reticuline to salutaridine, in accordance with embodimentsof the invention. FIG. 14A illustrates chromatogram traces showingreticuline and salutaridine for two epimerase variants (CYP-COR_89405,CYP-COR_4328) and a standard. FIG. 14B illustrates the same chromatogramtraces for salutaridine in (A) as replotted to demonstrate co-elutionwith the standard.

FIG. 15 illustrates a chiral LC/MS-MS analysis of yeast strainsengineered to convert racemic norlaudanosoline to (R)-reticuline, inaccordance with embodiments of the invention.

FIG. 16A illustrates N-linked glycosylation status of heterologouslyexpressed salutaridine synthase.

FIG. 16B illustrates engineered fusions of salutaridine synthase thateliminates N-linked glycosylation of the protein observed whenheterologously expressed in yeast but not plants, in accordance withembodiments of the invention

FIG. 17A and FIG. 17B illustrate cheilanthifoline synthase-salutaridinesynthase fusion designs (SEQ ID NOS 117, and 115-116, respectively, inorder of appearance), in accordance with embodiments of the invention.

FIG. 18 illustrates salutaridine synthase codon-optimization andengineered fusions that improve activity in yeast, in accordance withembodiments of the invention.

FIG. 19A and FIG. 19B illustrate LC/MS-MS analyses of small scale batchfermentation in which engineered yeast catalyze the conversion of(R)-reticuline to thebaine and the conversion of rac-norlaudanosoline tothebaine, in accordance with embodiments of the invention.

FIG. 20 illustrates generation of a CODM enzyme variant exhibitingenhanced activity in yeast through random mutagenesis and screening, inaccordance embodiments of the invention.

FIG. 21A, FIG. 21B, and FIG. 21C illustrate fermentation optimizationfor conversion of (R)-reticuline to thebaine by engineered yeast, inaccordance with embodiments of the invention.

FIG. 22 illustrates yeast platform strains for the production of the keybranch point intermediate reticuline from L-tyrosine, in accordance withembodiments of the invention.

FIG. 23 illustrates an enzyme having opioid 3-O-demethylase activity, inaccordance with embodiments of the invention.

FIG. 24 illustrates an enzyme having opioid N-demethylase activity, inaccordance with embodiments of the invention.

FIG. 25 illustrates an enzyme having N-methyltransferase activity, inaccordance with embodiments of the invention.

FIG. 26 illustrates a biosynthesis scheme in a microbial cell, inaccordance with embodiments of the invention.

FIG. 27A, FIG. 27B, and FIG. 27C illustrates the functional expressionof BM3 variants, in accordance with embodiments of the invention.

FIG. 28 illustrates plasmid/YAC vectors for enzyme expression andengineering, in accordance with embodiments of the invention.

FIG. 29 illustrates the functional expression of CODM, in accordancewith embodiments of the invention.

FIG. 30 illustrates a biosynthetic scheme for production ofsemi-synthetic opioids, in accordance with embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Nal-opioids are an important class of pharmacotherapies for combatingopioid addiction and opioid-associated side effects. Opiates likecodeine and morphine are plant molecules from opium poppy, which have aunique five-ring structure that allows them to bind μ-opioid receptorsin the brain, spine, gut, and peripheral sensors and mimic the body'snatural attenuation of pain. These same opiate molecules can be modifiedto act as antagonists, e.g., naltrexone and naloxone. In some examplesopioid molecules can be modified by introducing chemical modificationsthat permit binding to opioid receptors but prevent activation of thedownstream signaling response. Other molecules, such as buprenorphine,may bind to opioid receptors and act as mixed partial agonists.

The suite of antagonist and mixed partial-agonist opioids, collectivelycalled nal-opioids, may form a toolkit of competitive modulators thatcan occupy receptor binding sites in patients that have ingested astrong opioid agonist. As examples, in the addicted patient populationnal-opioids may be used for: (1) treating overdose, by administering astrong antagonist, such as naloxone; (2) detoxification, by managingsymptoms with mixed partial agonists, such as buprenorphine; and/or (3)maintenance, by blocking the reward response, for example with abuprenorphine/naloxone combination drug. In the patient population withsevere pain, nal-opioids may be used for: (4) prevention, e.g. throughabuse deterrent combination agonist/antagonist formulations, such asmorphine/naltrexone combinations, which may block euphoria andintoxication when the drug is misused; and/or (5) reducing side effectsby administering peripherally acting antagonists that may displaceopioid agonists from receptors in the gut where they may causeconstipation, for example, a polymer conjugate of naloxone (Movantik™).

The raw starting materials for nal-opioid synthesis are natural opiates,such as thebaine, that are extracted from opium poppy drug crops.Traditionally, these molecules are then chemically modified to thesemi-synthetic antagonists, weak agonists, and mixed partial agoniststhrough a series of inefficient reaction steps that require the use ofcatalysts, solvents, reagents, and purification methods to isolate thenal-opioid product from the starting material and reactionintermediates. The current semi-synthetic production methods areinefficient and add substantial cost to the overall process.

The present disclosure provides methods for the production of diversenal-opioids in engineered host cells. The present disclosure alsoprovides methods for the production of diverse nor-opioids in engineeredhost cells. Additionally, the present disclosure provides methods forthe production of an O-demethylase and an N-demethylase in engineeredhost cells. In particular cases, the disclosure provides methods forproducing nor-opioid products through the demethylization of an opioidto a nor-opioid in an engineered host cell. In further particular cases,the present disclosure provides methods for producing diversenal-opioids by modifying a nor-opioid with an enzyme that can add anN-linked side chain, such as an N-methyltransferase.

The present disclosure provides methods for the production ofnal-opioids and nor-opioid compounds in engineered host cells.Throughout this disclosure the term “compound” may be used to refer tosomething comprising two or more elements, for example a nal-opioidmolecule, or a nal-opioid composition. A nal-opioid compound may referto a largely pure composition of a nal-opioid, or a composition of anal-opioid which may or may not contain impurities.

Nal-Opioids of Interest

Host cells which produce BIAs of interest are provided. In someexamples, engineered strains of host cells such as the engineeredstrains of embodiments discussed herein may provide a platform forproducing Nal-opioids of interest including, but not limited to:naltrexone, naloxone, nalmefene, nalorphine, nalorphine, nalodeine,naldemedine, naloxegol, 6β-naltrexol, naltrindole, methylnaltrexone,methylsamidorphan, alvimopan, axelopran, bevenpran, dinicotinate,levallorphan, samidorphan, buprenorphine, dezocine, eptazocine,butorphanol, levorphanol, nalbuphine, pentazocine, phenazocine,norbinaltorphimine, and diprenorphine.

Nor-Opioids of Interest

Host cells which produce nor-opioids of interest are provided. In someexamples, engineered strains of host cells such as the engineeredstrains of embodiments discussed herein may provide a platform forproducing Nor-opioids of interest including, but not limited to:norcodeine, noroxycodone, northebaine, norhydrocodone,nordihydro-codeine, nor-14-hydroxy-codeine, norcodeinone,nor-14-hydroxy-codeinone, normorphine, noroxymorphone, nororipavine,norhydro-morphone, nordihydro-morphine, nor-14-hydroxy-morphine,normorphinone, nor-14-hydroxy-morphinone.

Benzylisoquinoline Alkaloids (BIAs) of Interest

Host cells which produce BIAs of interest are provided. In someexamples, engineered strains of host cells such as the engineeredstrains of embodiments discussed herein may provide a platform forproducing benzylisoquinoline alkaloids of interest and modificationsthereof across several structural classes including, but not limited to,precursor BIAs, benzylisoquinolines, promorphinans, morphinans andothers. Each of these classes is meant to include biosyntheticprecursors, intermediates, and metabolites thereof, of any convenientmember of an engineered host cell biosynthetic pathway that may lead toa member of the class. Non-limiting examples of compounds are givenbelow for each of these structural classes. In some cases, the structureof a given example may or may not be characterized itself as abenzylisoquinoline alkaloid. In some cases, the present chemicalentities are meant to include all possible isomers, including singleenantiomers, racemic mixtures, optically pure forms, mixtures ofdiastereomers, and intermediate mixtures.

BIA precursors may include, but are not limited to, norcoclaurine (NC)and norlaudanosoline (NL), as well as NC and NL precursors, such astyrosine, tyramine, 4-hydroxyphenylacetaldehyde (4-HPA),4-hydroxyphenylpyruvic acid (4-HPPA), L-3,4-dihydroxyphenylalanine(L-DOPA), 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA), and dopamine. Insome embodiments, the one or more BIA precursors are3,4-dihydroxyphenylacetaldehyde (3,4-DHPA) and dopamine. In certaininstances, the one or more BIA precursors are4-hydroxyphenylacetaldehyde (4-HPA) and dopamine. In particular, NL andNC may be synthesized, respectively, from precursor molecules via aPictet-Spengler condensation reaction, where the reaction may occurspontaneously or may by catalyzed by any convenient enzymes.

Benzylisoquinolines may include, but are not limited to, norcoclaurine,norlaudanosoline, coclaurine, 3′-hydroxycoclaurine,4′-O-methylnorlaudanosoline, 4′-O-methyl-laudanosoline,N-methylnorcoclaurine, laudanosoline, N-methylcoclaurine,3′-hydroxy-N-methylcoclaurine, reticuline, norreticuline, papaverine,laudanine, laudanosine, tetrahydropapaverine, 1,2-dihydropapaverine, andorientaline.

Promorphinans may include, but are not limited to, salutaridine,salutaridinol, and salutaridinol-7-O-acetate.

Morphinans may include, but are not limited to, thebaine, codeinone,codeine, morphine, morphinone, oripavine, neopinone, neopine,neomorphine, hydrocodone, dihydrocodeine, 14-hydroxycodeinone,oxycodone, 14-hydroxycodeine, morphinone, hydromorphone,dihydromorphine, dihydroetorphine, ethylmorphine, etorphine, metopon,buprenorphine, pholcodine, and heterocodeine.

Host Cells

Any convenient cells may be utilized in the subject host cells andmethods. In some cases, the host cells are non-plant cells. In someinstances, the host cells may be characterized as microbial cells. Incertain cases, the host cells are insect cells, vertebrate cells,mammalian cells, plant cells, fungal cells, bacterial cells, or yeastcells. Any convenient type of host cell may be utilized in producing thesubject nor-opioid- or nal-opioid-producing cells, see, e.g.,US2008/0176754, and US2014/0273109 the disclosures of which areincorporated by reference in their entirety. Host cells of interestinclude, but are not limited to, bacterial cells, such as Bacillussubtilis, Escherichia coli, Streptomyces, and Salmonella typhimuiumcells, insect cells such as Drosophila melanogaster S2 and Spodopterafrugiperda Sf9 cells, mammalian cells such as HeLa and 293 cells, plantcells such as Tobacco BY-2 cells, and yeast cells such as Saccharomycescerevisiae, Schizosaccharomyces pombe, and Pichia pastoris cells. Insome examples, the host cells are yeast cells or E. coli cells. In somecases, the host cell is a yeast cell. In some instances, the host cellis from a strain of yeast engineered to produce a nor-opioid ornal-opioid BIA of interest, such as a northebaine or naloxone. In someinstances, the host cell is from a strain of yeast engineered to producean enzyme of interest. In some instances, the host cell is from a strainof yeast engineered to produce an O-demethylase. The O-demethylase maybe able to convert a substrate, such as a first opioid, into a secondopioid. In some instances, the host cell is from a strain of yeastengineered to produce an N-demethylase. The N-demethylase may be able toconvert a substrate, such as a second opioid, into a nor-opioid. In someinstances, the host cell is from a strain of yeast engineered to producea methyltransferase. Additionally the methyltransferase may be able toconvert a nor-opioid into a nal-opioid. In some instances, the host cellis from a strain of yeast engineered to produce an epimerase. Theepimerase may have an oxidase and a reductase. Additionally, theepimerase may be able to convert an (S)-1-benzylisoquinoline alkaloid toan (R)-1-benzylisoquinoline alkaloid. Further, the epimerase may beseparated into smaller enzymes that retain oxidase or reductase activityso as to be used to convert an (S)-1-benzylisoquinoline alkaloid to an(R)-1-benzylisoquinoline alkaloid.

Any of the host cells described in US2008/0176754 and US2014/0273109 bySmolke et al. may be adapted for use in the subject cells and methods.In certain embodiments, the yeast cells may be of the speciesSaccharomyces cerevisiae (S. cerevisiae). In certain embodiments, theyeast cells may be of the species Schizosaccharomyces pombe. In certainembodiments, the yeast cells may be of the species Pichia pastoris.Yeast is of interest as a host cell because cytochrome P450 proteins areable to fold properly into the endoplasmic reticulum membrane so thattheir activity is maintained. In examples, cytochrome P450 proteins areinvolved in some biosynthetic pathways of interest. In additionalexamples, cytochrome P450 proteins are involved in the production ofBIAs of interest such as naloxone or naltrexone. In further examples,cytochrome P450 proteins are involved in the production of an enzyme ofinterest, such as an epimerase having an oxidase and a reductase.

Yeast strains of interest that may find use in the invention include,but are not limited to, CEN.PK (Genotype: MATa/α ura3-52/ura3-52trp1-289/trp1-289 leu2-3_112/leu2-3_112 his3 Δ1/his3 Δ1 MAL2-8C/MAL2-8CSUC2/SUC2), S288C, W303, D273-10B, X2180, A364A, Σ1278B, AB972, SK1, andFL100. In certain cases, the yeast strain is any of S288C (MATα; SUC2mal mel gal2 CUP1 flo1 flo8-1 hap1), BY4741 (MATα; his3Δ1; leu2Δ0;met15Δ0; ura3Δ0), BY4742 (MATα; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0), BY4743(MATa/MATα; his3Δ1/his3Δ1; leu2Δ0/leu2Δ0; met15Δ0/MET15; LYS2/lys2Δ0;ura3Δ0/ura3Δ0), and WAT11 or W(R), derivatives of the W303-B strain(MATa; ade2-1; his3-11, -15; leu2-3, -112; ura3-1; canR; cyr+) whichexpress the Arabidopsis thaliana NADPH-P450 reductase ATR1 and the yeastNADPH-P450 reductase CPR1, respectively. In another embodiment, theyeast cell is W303alpha (MATa; his3-11, 15 trp1-1 leu2-3 ura3-1 ade2-1).The identity and genotype of additional yeast strains of interest may befound at EUROSCARF(web.uni-frankfurt.de/fb15/mikro/euroscarf/col_index.html).

In some instances the host cell is a fungal cell. In certainembodiments, the fungal cells may be of the Aspergillus species andstrains include Aspergillus niger (ATCC 1015, ATCC 9029, CBS 513.88),Aspergillus oryzae (ATCC 56747, RIB40), Aspergillus terreus (NIH 2624,ATCC 20542) and Aspergillus nidulans (FGSC A4).

In certain embodiments, heterologous coding sequences may be codonoptimized for expression in Aspergillus sp. and expressed from anappropriate promoter. In certain embodiments, the promoter may beselected from phosphoglycerate kinase promoter (PGK), MbfA promoter,cytochrome c oxidase subunit promoter (CoxA), SrpB promoter, TvdApromoter, malate dehydrogenase promoter (MdhA), beta-mannosidasepromoter (ManB). In certain embodiments, a terminator may be selectedfrom glucoamylase terminator (GlaA) or TrpC terminator. In certainembodiments, the expression cassette consisting of a promoter,heterologous coding sequence, and terminator may be expressed from aplasmid or integrated into the genome of the host. In certainembodiments, selection of cells maintaining the plasmid or integrationcassette may be performed with antibiotic selection such as hygromycinor nitrogen source utilization, such as using acetamide as a solenitrogen source. In certain embodiments, DNA constructs may beintroduced into the host cells using established transformation methodssuch as protoplast transformation, lithium acetate, or electroporation.In certain embodiments, cells may be cultured in liquid ME or solid MEA(3% malt extract, 0.5% peptone, and ±1.5% agar) or in Vogel's minimalmedium with or without selection.

In some instances the host cell is a bacterial cell. The bacterial cellmay be selected from any bacterial genus. Examples of genuses from whichthe bacterial cell may come include Anabaena, Arthrobacter, Acetobacter,Acetobacterium, Bacillus, Bifidobacterium, Brachybacterium,Brevibacterium, Carnobacterium, Clostridium, Corynebacterium,Enterobacter, Escherichia, Gluconacetobacter, Gluconobacter, Hafnia,Halomonas, Klebsiella, Kocuria, Lactobacillus, Leucononstoc,Macrococcus, Methylomonas, Methylobacter, Methylocella, Methylococcus,Microbacterium, Micrococcus, Microcystis, Moorella, Oenococcus,Pediococcus, Prochlorococcus, Propionibacterium, Proteus,Pseudoalteromonas, Pseudomonas, Psychrobacter, Rhodobacter, Rhodococcus,Rhodopseudomonas, Serratia, Staphylococcus, Streptococcus, Streptomyces,Synechococcus, Synechocystis, Tetragenococcus, Weissella, and Zymomonas.Examples of bacterial species which may be used with the methods of thisdisclosure include Arthrobacter nicotianae, Acetobacter aceti,Arthrobacter arilaitensis, Bacillus cereus, Bacillus coagulans, Bacilluslicheniformis, Bacillus pumilus, Bacillus sphaericus, Bacillusstearothermophilus, Bacillus subtilis, Bifidobacterium adolescentis,Brachybacterium tyrofermentans, Brevibacterium linens, Carnobacteriumdivergens, Corynebacterium flavescens, Enterococcus faecium,Gluconacetobacter europaeus, Gluconacetobacter johannae, Gluconobacteroxydans, Hafnia alvei, Halomonas elongata, Kocuria rhizophila,Lactobacillus acidifarinae, Lactobacillus jensenii, Lactococcus lactis,Lactobacillus yamanashiensis, Leuconostoc citreum, Macrococcuscaseolyticus, Microbacterium foliorum, Micrococcus lylae, Oenococcusoeni, Pediococcus acidilactici, Propionibacterium acidipropionici,Proteus vulgaris, Pseudomonas fluorescens, Psychrobacter celer,Staphylococcus condimenti, Streptococcus thermophilus, Streptomycesgriseus, Tetragenococcus halophilus, Weissella cibaria, Weissellakoreensis, Zymomonas mobilis, Corynebacterium glutamicum,Bifidobacterium bifidum/breve/longum, Streptomyces lividans,Streptomyces coelicolor, Lactobacillus plantarum, Lactobacillus sakei,Lactobacillus casei, Pseudoalteromonas citrea, Pseudomonas putida,Clostridium ljungdahlii/aceticum/acetobutylicum/beijerinckii/butyricum,and Moorella themocellum/thermoacetica.

In certain embodiments, the bacterial cells may be of a strain ofEscherichia coli. In certain embodiments, the strain of E. coli may beselected from BL21, DH5α, XL1-Blue, HB101, BL21, and K12, In certainembodiments, heterologous coding sequences may be codon optimized forexpression in E. coli and expressed from an appropriate promoter. Incertain embodiments, the promoter may be selected from T7 promoter, tacpromoter, trc promoter, tetracycline-inducible promoter (tet), lacoperon promoter (lac), lacO1 promoter. In certain embodiments, theexpression cassette consisting of a promoter, heterologous codingsequence, and terminator may be expressed from a plasmid or integratedinto the genome. In certain embodiments, the plasmid is selected frompUC19 or pBAD. In certain embodiments, selection of cells maintainingthe plasmid or integration cassette may be performed with antibioticselection such as kanamycin, chloramphenicol, streptomycin,spectinomycin, gentamycin, erythromycin or ampicillin. In certainembodiments, DNA constructs may be introduced into the host cells usingestablished transformation methods such as conjugation, heat shockchemical transformation, or electroporation. In certain embodiments,cells may be cultured in liquid Luria-Bertani (LB) media at 37° C. withor without antibiotics.

In certain embodiments, the bacterial cells may be a strain of Bacillussubtilis. In certain embodiments, the strain of B. subtilis may beselected from 1779, GP25, RO-NN-1, 168, BSn5, BEST195, 1A382, and 62178.In certain embodiments, heterologous coding sequences may be codonoptimized for expression in Bacillus sp. and expressed from anappropriate promoter. In certain embodiments, the promoter may beselected from grac promoter, p43 promoter, or trnQ promoter. In certainembodiments, the expression cassette consisting of the promoter,heterologous coding sequence, and terminator may be expressed from aplasmid or integrated into the genome. In certain embodiments, theplasmid is selected from pHP13 pE194, pC194, pHT01, or pHT43. In certainembodiments, integrating vectors such as pDG364 or pDG1730 may be usedto integrate the expression cassette into the genome. In certainembodiments, selection of cells maintaining the plasmid or integrationcassette may be performed with antibiotic selection such aserythromycin, kanamycin, tetracycline, and spectinomycin. In certainembodiments, DNA constructs may be introduced into the host cells usingestablished transformation methods such as natural competence, heatshock, or chemical transformation. In certain embodiments, cells may becultured in liquid Luria-Bertani (LB) media at 37° C. or M9 medium plusglucose and tryptophan.

Genetic Modifications to Host Cells

The host cells may be engineered to include one or more modifications(such as two or more, three or more, four or more, five or more, or evenmore modifications) that provide for the production of BIAs of interest.Additionally or alternatively, the host cells may be engineered toinclude one or more modifications (such as two or more, three or more,four or more, five or more, or even more modifications) that provide forthe production of enzymes of interest. In some cases, a modification isa genetic modification, such as a mutation, addition, or deletion of agene or fragment thereof, or transcription regulation of a gene orfragment thereof. As used herein, the term “mutation” refers to adeletion, insertion, or substitution of an amino acid(s) residue ornucleotide(s) residue relative to a reference sequence or motif. Themutation may be incorporated as a directed mutation to the native geneat the original locus. In some cases, the mutation may be incorporatedas an additional copy of the gene introduced as a genetic integration ata separate locus, or as an additional copy on an episomal vector such asa 2μ or centromeric plasmid. In certain instances, the substrateinhibited copy of the enzyme is under the native cell transcriptionalregulation. In some instances, the substrate inhibited copy of theenzyme is introduced with engineered constitutive or dynamic regulationof protein expression by placing it under the control of a syntheticpromoter. In some examples, the object of one or more modifications maybe a native gene. In some examples, the object of one or moremodifications may be a non-native gene. In some examples, a non-nativegene may be inserted into a host cell. In further examples, a non-nativegene may be altered by one or more modifications prior to being insertedinto a host cell.

An engineered host cell may overproduce one or more nor-opioid BIAs ofinterest. An engineered host cell may overproduce one or more nal-opioidBIAs of interest. By overproduce is meant that the cell has an improvedor increased production of a nor-opioid and/or nal-opioid BIA moleculeof interest relative to a control cell (e.g., an unmodified cell). Byimproved or increased production is meant both the production of someamount of the nor-opioid and/or nal-opioid BIA of interest where thecontrol has no nor-opioid and/or nal-opioid BIA of interest production,as well as an increase of about 10% or more, such as about 20% or more,about 30% or more, about 40% or more, about 50% or more, about 60% ormore, about 80% or more, about 100% or more, such as 2-fold or more,such as 5-fold or more, including 10-fold or more in situations wherethe control has some nor-opioid and/or nal-opioid BIA of interestproduction.

An engineered host cell may overproduce one or more nor-opioids. In somecases, the engineered host cell may produce some amount of thenor-opioid of interest where the control has no nor-opioid production,as well as an increase of about 10% or more, such as about 20% or more,about 30% or more, about 40% or more, about 50% or more, about 60% ormore, about 80% or more, about 100% or more, such as 2-fold or more,such as 5-fold or more, including 10-fold or more in situations wherethe control has some nor-opioid of interest production.

An engineered host cell may further overproduce one or more nal-opioids.In some cases, the engineered host cell may produce some amount of thenal-opioid of interest where the control has no nal-opioid production,as well as an increase of about 10% or more, such as about 20% or more,about 30% or more, about 40% or more, about 50% or more, about 60% ormore, about 80% or more, about 100% or more, such as 2-fold or more,such as 5-fold or more, including 10-fold or more in situations wherethe control has some nal-opioid of interest production.

An engineered host cell may overproduce one or more BIAs of interest. Byoverproduce is meant that the cell has an improved or increasedproduction of a BIA molecule of interest relative to a control cell(e.g., an unmodified cell). By improved or increased production is meantboth the production of some amount of the BIA of interest where thecontrol has no BIA of interest production, as well as an increase ofabout 10% or more, such as about 20% or more, about 30% or more, about40% or more, about 50% or more, about 60% or more, about 80% or more,about 100% or more, such as 2-fold or more, such as 5-fold or more,including 10-fold or more in situations where the control has some BIAof interest production.

An engineered host cell may overproduce one or more(S)-1-benzylisoquinoline alkaloids. In some cases, the engineered hostcell may produce some amount of the (S)-1-benzylisoquinoline alkaloid ofinterest where the control has no (S)-1-benzylisoquinoline alkaloidproduction, as well as an increase of about 10% or more, such as about20% or more, about 30% or more, about 40% or more, about 50% or more,about 60% or more, about 80% or more, about 100% or more, such as 2-foldor more, such as 5-fold or more, including 10-fold or more in situationswhere the control has some (S)-1-benzylisoquinoline alkaloid of interestproduction.

An engineered host cell may further overproduce one or more(R)-1-benzylisoquinoline alkaloids. In some cases, the engineered hostcell may produce some amount of the (R)-1-benzylisoquinoline alkaloid ofinterest where the control has no (R)-1-benzylisoquinoline alkaloidproduction, as well as an increase of about 10% or more, such as about20% or more, about 30% or more, about 40% or more, about 50% or more,about 60% or more, about 80% or more, about 100% or more, such as 2-foldor more, such as 5-fold or more, including 10-fold or more in situationswhere the control has some (R)-1-benzylisoquinoline alkaloid of interestproduction. An engineered host cell may further overproduce one or moreof morphinan and pro-morphinan alkaloids.

In some cases, the engineered host cell is capable of producing anincreased amount of (R)-reticuline relative to a control host cell thatlacks the one or more modifications (e.g., as described herein). Incertain instances, the increased amount of (R)-reticuline is about 10%or more relative to the control host cell, such as about 20% or more,about 30% or more, about 40% or more, about 50% or more, about 60% ormore, about 80% or more, about 100% or more, about 2-fold or more, about5-fold or more, or even about 10-fold or more relative to the controlhost cell. In some cases, (R)-reticuline is the product of anepimerization reaction within an engineered host cell. In these cases,(S)-reticuline may be the substrate of the epimerization reaction.

Additionally, an engineered host cell may overproduce one or moreenzymes of interest. By overproduce is meant that the cell has animproved or increased production of an enzyme of interest relative to acontrol cell (e.g., an unmodified cell). By improved or increasedproduction is meant both the production of some amount of the enzyme ofinterest where the control has no production, as well as an increase ofabout 10% or more, such as about 20% or more, about 30% or more, about40% or more, about 50% or more, about 60% or more, about 80% or more,about 100% or more, such as 2-fold or more, such as 5-fold or more,including 10-fold or more in situations where the control has someenzyme of interest production.

An engineered host cell may overproduce one or more O-demethylase (ODM)enzymes. Examples of ODM enzymes that may be utilized in embodimentsdescribed herein are found in Table 3. In some cases, the engineeredhost cell may produce some amount of the ODM enzyme where the controlhas no ODM enzyme production, as well as an increase of about 10% ormore, such as about 20% or more, about 30% or more, about 40% or more,about 50% or more, about 60% or more, about 80% or more, about 100% ormore, such as 2-fold or more, such as 5-fold or more, including 10-foldor more in situations where the control has some ODM enzyme production.

An engineered host cell may overproduce one or more N-demethylase (NDM)enzymes. Examples of NDM enzymes that may be utilized in embodimentsdescribed herein are found in Table 4. In some cases, the engineeredhost cell may produce some amount of the ODM enzyme where the controlhas no NDM enzyme production, as well as an increase of about 10% ormore, such as about 20% or more, about 30% or more, about 40% or more,about 50% or more, about 60% or more, about 80% or more, about 100% ormore, such as 2-fold or more, such as 5-fold or more, including 10-foldor more in situations where the control has some NDM enzyme production.

An engineered host cell may overproduce one or more N-methyltransferase(NMT) enzymes. Examples of NMT enzymes that may be utilized inembodiments described herein are found in Table 5. In some cases, theengineered host cell may produce some amount of the NMT enzyme where thecontrol has no NMT enzyme production, as well as an increase of about10% or more, such as about 20% or more, about 30% or more, about 40% ormore, about 50% or more, about 60% or more, about 80% or more, about100% or more, such as 2-fold or more, such as 5-fold or more, including10-fold or more in situations where the control has some NMT enzymeproduction.

An engineered host cell may overproduce one or more CYP-COR enzymes. Insome cases, the engineered host cell may produce some amount of theCYP-COR enzyme where the control has no CYP-COR enzyme production, aswell as an increase of about 10% or more, such as about 20% or more,about 30% or more, about 40% or more, about 50% or more, about 60% ormore, about 80% or more, about 100% or more, such as 2-fold or more,such as 5-fold or more, including 10-fold or more in situations wherethe control has some CYP-COR enzyme production.

An engineered host cell may further overproduce one or more enzymes thatare derived from the CYP-COR enzyme. In some cases, the engineered hostcell may produce some amount of the enzymes that are derived from theCYP-COR enzyme, where the control has no production of enzymes that arederived from the CYP-COR enzyme, as well as an increase of about 10% ormore, such as about 20% or more, about 30% or more, about 40% or more,about 50% or more, about 60% or more, about 80% or more, about 100% ormore, such as 2-fold or more, such as 5-fold or more, including 10-foldor more in situations where the control has some production of enzymesthat are derived from the CYP-COR enzyme.

In some cases, the one or more (such as two or more, three or more, orfour or more) modifications may be selected from: a substrate inhibitionalleviating mutation in a biosynthetic enzyme gene; a product inhibitionalleviating mutation in a biosynthetic enzyme gene; a cofactor recoverypromoting mechanism; a feedback inhibition alleviating mutation in abiosynthetic enzyme gene; a transcriptional modulation modification of abiosynthetic enzyme gene; an inactivating mutation in an enzyme gene; anepimerization modification; a bisBIA generating modification; and aheterologous coding sequence that encodes an enzyme. A cell thatincludes one or more modifications may be referred to as an engineeredcell.

Substrate Inhibition Alleviating Mutations

In some instances, the engineered host cells are cells that include oneor more substrate inhibition alleviating mutations (such as two or more,three or more, four or more, five or more, or even more) in one or morebiosynthetic enzyme genes of the cell. In some examples, the one or morebiosynthetic enzyme genes are native to the cell (e.g., is present in anunmodified cell). In some examples, the one or more biosynthetic enzymegenes are non-native to the cell. As used herein, the term “substrateinhibition alleviating mutation” refers to a mutation that alleviates asubstrate inhibition control mechanism of the cell.

A mutation that alleviates substrate inhibition reduces the inhibitionof a regulated enzyme in the cell of interest relative to a control celland provides for an increased level of the regulated compound or adownstream biosynthetic product thereof. In some cases, by alleviatinginhibition of the regulated enzyme is meant that the IC₅₀ of inhibitionis increased by 2-fold or more, such as by 3-fold or more, 5-fold ormore, 10-fold or more, 30-fold or more, 100-fold or more, 300-fold ormore, 1000-fold or more, or even more. By increased level is meant alevel that is 110% or more of that of the regulated compound in acontrol cell or a downstream product thereof, such as 120% or more, 130%or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% ormore, 190% or more, or 200% or more, such as at least 3-fold or more, atleast 5-fold or more, at least 10-fold or more or even more of theregulated compound in the engineered host cell or a downstream productthereof.

A variety of substrate inhibition control mechanisms and biosyntheticenzymes in the engineered host cell that are directed to regulation oflevels of nal-opioid or nor-opioid BIAs of interest, or precursorsthereof, may be targeted for substrate inhibition alleviation. Theengineered host cell may include one or more substrate inhibitionalleviating mutations in one or more biosynthetic enzyme genes. The oneor more mutations may be located in any convenient biosynthetic enzymegenes where the biosynthetic enzyme is subject to regulatory control. Insome embodiments, the one or more biosynthetic enzyme genes encode oneor more tyrosine hydroxylase enzymes. In certain instances, the one ormore substrate inhibition alleviating mutations are present in abiosynthetic enzyme gene that is TyrH. In some embodiments, theengineered host cell may include one or more substrate inhibitionalleviating mutations in one or more biosynthetic enzyme genes such asone of those genes described in Table 2.

In certain embodiments, the one or more substrate inhibition alleviatingmutations are present in the TyrH gene. The TyrH gene encodes tyrosinehydroxylase, which is an enzyme that converts tyrosine to L-DOPA.However, TyrH is inhibited by its substrate, tyrosine. Mammaliantyrosine hydroxylase activity, such as that seen in humans or rats, canbe improved through mutations to the TyrH gene that relieve substrateinhibition. In particular, substrate inhibition from tyrosine can berelieved by a point mutation W166Y in the TyrH gene. The point mutationW166Y in the TyrH gene may also improve the binding of the cosubstrateof tyrosine hydroxylase, BH₄, to catalyze the reaction of tyrosine toL-DOPA. The mutants of TyrH, when expressed in yeast strains to produceBIAs from sugar (such as those described in U.S. Provisional PatentApplication Ser. No. 61/899,496) can significantly improve theproduction of BIAs.

Any convenient numbers and types of mutations may be utilized toalleviate a substrate inhibition control mechanism. In certainembodiments, the engineered host cells of the present invention mayinclude 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 ormore, 13 or more, 14 or more, or even 15 or more substrate inhibitionalleviating mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 substrate inhibition alleviating mutations in one or morebiosynthetic enzyme genes within the engineered host cell.

Cofactor Recovery Promoting Mechanisms

In some instances, the engineered host cells are cells that include oneor more cofactor recovery promoting mechanisms (such as two or more,three or more, four or more, five or more, or even more) in one or morebiosynthetic enzyme genes of the cell. In some examples, the one or morebiosynthetic enzyme genes are native to the cell (e.g., is present in anunmodified cell). In some examples, the one or more biosynthetic enzymegenes are non-native to the cell. As used herein, the term “cofactorrecovery promoting mechanism” refers to a mechanism that promotes acofactor recovery control mechanism of the cell.

A variety of cofactor recovery control mechanisms and biosyntheticenzymes in the engineered host cell that are directed to regulation oflevels of nor-opioid or nal-opioid BIAs of interest, or precursorsthereof, may be targeted for cofactor recovery promotion. The engineeredhost cell may include one or more cofactor recovery promoting mechanismin one or more biosynthetic enzyme genes. In examples, the engineeredhost cell may include a heterologous coding sequence that encodesdihydrofolate reductase (DHFR). When DHFR is expressed, it may convert7,8-dihydrobiopterin (BH₂) to the tetrahydrobiopterin (BH₄), therebyrecovering BH₄ as a TyrH cosubstrate. In some examples, the engineeredhost cell may include one or more cofactor recovery promoting mechanismsin one or more biosynthetic enzyme genes such as one of those genesdescribed in Table 2.

Any convenient numbers and types of mechanisms may be utilized topromote a cofactor recovery control mechanism. In certain embodiments,the engineered host cells of the present invention may include 1 ormore, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more,8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14or more, or even 15 or more cofactor recovery promoting mechanisms suchas 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 cofactor recoverypromoting mechanisms in one or more biosynthetic enzyme genes within theengineered host cell.

Product Inhibition Alleviating Mutations

In some instances, the engineered host cells are cells that include oneor more product inhibition alleviating mutations (such as two or more,three or more, four or more, five or more, or even more) in one or morebiosynthetic enzyme genes of the cell. In some examples, the one or morebiosynthetic enzyme genes are native to the cell (e.g., is present in anunmodified cell). In some examples, the one or more biosynthetic enzymegenes are non-native to the cell. As used herein, the term “productinhibition alleviating mutation” refers to a mutation that alleviates ashort term and/or long term product inhibition control mechanism of anengineered host cell. Short term product inhibition is a controlmechanism of the cell in which there is competitive binding at acosubstrate binding site. Long term product inhibition is a controlmechanism of the cell in which there is irreversible binding of acompound away from a desired pathway.

A mutation that alleviates product inhibition reduces the inhibition ofa regulated enzyme in the cell of interest relative to a control celland provides for an increased level of the regulated compound or adownstream biosynthetic product thereof. In some cases, by alleviatinginhibition of the regulated enzyme is meant that the IC₅₀ of inhibitionis increased by 2-fold or more, such as by 3-fold or more, 5-fold ormore, 10-fold or more, 30-fold or more, 100-fold or more, 300-fold ormore, 1000-fold or more, or even more. By increased level is meant alevel that is 110% or more of that of the regulated compound in acontrol cell or a downstream product thereof, such as 120% or more, 130%or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% ormore, 190% or more, or 200% or more, such as at least 3-fold or more, atleast 5-fold or more, at least 10-fold or more or even more of theregulated compound in the engineered host cell or a downstream productthereof.

A variety of product inhibition control mechanisms and biosyntheticenzymes in the engineered host cell that are directed to regulation oflevels of nor-opioid or nal-opioid BIAs of interest may be targeted forproduct inhibition alleviation. The engineered host cell may include oneor more product inhibition alleviating mutations in one or morebiosynthetic enzyme genes. The mutation may be located in any convenientbiosynthetic enzyme genes where the biosynthetic enzyme is subject toregulatory control. In some embodiments, the one or more biosyntheticenzyme genes encode one or more tyrosine hydroxylase enzymes. In certaininstances, the one or more product inhibition alleviating mutations arepresent in a biosynthetic enzyme gene that is TyrH. In some embodiments,the engineered host cell includes one or more product inhibitionalleviating mutations in one or more biosynthetic enzyme genes such asone of those genes described in Table 2.

In certain embodiments, the one or more product inhibition alleviatingmutations are present in the TyrH gene. The TyrH gene encodes tyrosinehydroxylase, which is an enzyme that converts tyrosine to L-DOPA. TyrHrequires tetrahydrobiopterin (BH₄) as a cosubstrate to catalyze thehydroxylation reaction. Some microbial strains, such as Saccharomycescerevisiae, do not naturally produce BH₄, but can be engineered toproduce this substrate through a four-enzyme synthesis and recyclingpathway, as illustrated in FIG. 1. FIG. 1 illustrates examples ofsynthesis, recycling, and salvage pathways of tetrahydrobiopterin, inaccordance with embodiments of the invention. FIG. 1 provides the use ofthe enzymes PTPS, pyruvoyl tetrahydropterin synthase; SepR, sepiapterinreductase; PCD, pterin 4a-carbinolamine dehydratase; QDHPR,dihydropteridine reductase; and DHFR, dihydrofolate reductase. Of theenzymes that are illustrated in FIG. 1, yeast synthesizes an endogenousGTP cyclohydrolase I. GTP and dihydroneopterin triphosphate arenaturally synthesized in yeast. Additionally, other metabolites in FIG.1 are not naturally produced in yeast.

TyrH is inhibited by its product L-DOPA, as well as othercatecholamines, particularly dopamine. Mammalian tyrosine hydroxylaseactivity, such as from humans or rats, can be improved through mutationsthat relieve product inhibition. For example, short term productinhibition, such as competitive binding at the cosubstrate binding site,can be relieved by a point mutation W166Y on the TyrH gene. Inparticular, the point mutation W166Y on the TyrH gene may improvebinding of the cosubstrate. Additionally, short term product inhibitionto relieve competitive binding at the cosubstrate binding site may beimproved by a point mutation S40D on the TyrH gene. Short term productinhibition may also be improved by the joint mutations of R37E, R38E onthe TyrH gene. In particular, R37E, R38E mutations may togetherspecifically improve tyrosine hydroxylase activity in the presence ofdopamine.

Additionally, long term product inhibition may be relieved by pointmutations on the TyrH gene. Long term product inhibition relief mayinclude the irreversible binding of catecholamine to iron in the activesite such that there is less catecholamine present to act as a productinhibitor of tyrosine hydroxylase activity. Long term product inhibitioncan be relieved by the mutations E332D and Y371F, respectively, in theTyrH gene.

Combinations of the mutations can be made (such as two or three or moremutations at once) to relieve multiple types of substrate and productinhibition to further improve the activity of TyrH. The mutants of TyrH,when expressed in yeast strains to produce nor-opioid and/or nal-opioidBIAs of interest from sugar (such as those described in U.S. ProvisionalPatent Application Ser. No. 61/899,496) can significantly improve theproduction of nor-opioid and/or nal-opioid products.

Any convenient numbers and types of mutations may be utilized toalleviate a product inhibition control mechanism. In certainembodiments, the engineered host cells of the present invention mayinclude 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 ormore, 13 or more, 14 or more, or even 15 or more product inhibitionalleviating mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 product inhibition alleviating mutations in one or morebiosynthetic enzyme genes within the engineered host cell.

Feedback Inhibition Alleviating Mutations

In some instances, the engineered host cells are cells that include oneor more feedback inhibition alleviating mutations (such as two or more,three or more, four or more, five or more, or even more) in one or morebiosynthetic enzyme genes of the cell. In some cases, the one or morebiosynthetic enzyme genes are native to the cell (e.g., is present in anunmodified cell). Additionally or alternatively, in some examples theone or more biosynthetic enzyme genes are non-native to the cell. Asused herein, the term “feedback inhibition alleviating mutation” refersto a mutation that alleviates a feedback inhibition control mechanism ofan engineered host cell. Feedback inhibition is a control mechanism ofthe cell in which an enzyme in the synthetic pathway of a regulatedcompound is inhibited when that compound has accumulated to a certainlevel, thereby balancing the amount of the compound in the cell. Amutation that alleviates feedback inhibition reduces the inhibition of aregulated enzyme in the engineered host cell relative to a control cell.In this way, engineered host cell provides for an increased level of theregulated compound or a downstream biosynthetic product thereof. In somecases, by alleviating inhibition of the regulated enzyme is meant thatthe IC₅₀ of inhibition is increased by 2-fold or more, such as by 3-foldor more, 5-fold or more, 10-fold or more, 30-fold or more, 100-fold ormore, 300-fold or more, 1000-fold or more, or even more. By increasedlevel is meant a level that is 110% or more of that of the regulatedcompound in a control cell or a downstream product thereof, such as 120%or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% ormore, 180% or more, 190% or more, or 200% or more, such as at least3-fold or more, at least 5-fold or more, at least 10-fold or more oreven more of the regulated compound in the host cell or a downstreamproduct thereof.

A variety of feedback inhibition control mechanisms and biosyntheticenzymes that are directed to regulation of levels of BIAs of interestmay be targeted for alleviation in the host cell. The host cell mayinclude one or more feedback inhibition alleviating mutations in one ormore biosynthetic enzyme genes native to the cell. The one or moremutations may be located in any convenient biosynthetic enzyme geneswhere the biosynthetic enzyme is subject to regulatory control. In someembodiments, the one or more biosynthetic enzyme genes may encode one ormore enzymes selected from a3-deoxy-d-arabinose-heptulosonate-7-phosphate (DAHP) synthase and achorismate mutase. In some embodiments, the one or more biosyntheticenzyme genes encode a 3-deoxy-d-arabinose-heptulosonate-7-phosphate(DAHP) synthase. In some instances, the one or more biosynthetic enzymegenes may encode a chorismate mutase. In certain instances, the one ormore feedback inhibition alleviating mutations may be present in abiosynthetic enzyme gene selected from ARO4 and ARO7. In certaininstances, the one or more feedback inhibition alleviating mutations maybe present in a biosynthetic enzyme gene that is ARO4. In certaininstances, the one or more feedback inhibition alleviating mutations arepresent in a biosynthetic enzyme gene that is ARO7. In some embodiments,the engineered host cell may include one or more feedback inhibitionalleviating mutations in one or more biosynthetic enzyme genes such asone of those genes described in Table 2.

Any convenient numbers and types of mutations may be utilized toalleviate a feedback inhibition control mechanism. As used herein, theterm “mutation” refers to a deletion, insertion, or substitution of anamino acid(s) residue or nucleotide(s) residue relative to a referencesequence or motif. The mutation may be incorporated as a directedmutation to the native gene at the original locus. In some cases, themutation may be incorporated as an additional copy of the geneintroduced as a genetic integration at a separate locus, or as anadditional copy on an episomal vector such as a 2μ or centromericplasmid. In certain instances, the feedback inhibited copy of the enzymeis under the native cell transcriptional regulation. In some instances,the feedback inhibited copy of the enzyme is introduced with engineeredconstitutive or dynamic regulation of protein expression by placing itunder the control of a synthetic promoter.

In certain embodiments, the one or more feedback inhibition alleviatingmutations may be present in the ARO4 gene. ARO4 mutations of interestmay include, but are not limited to, substitution of the lysine residueat position 229 with a leucine, a substitution of the glutamine residueat position 166 with a lysine residue, or a mutation as described byHartmann M, et al. ((2003) Proc Natl Acad Sci USA 100(3):862-867) orFukuda, et al. ((1992) J Ferment Bioeng 74(2):117-119). In someinstances, mutations for conferring feedback inhibition may be selectedfrom a mutagenized library of enzyme mutants. Examples of suchselections may include rescue of growth of o-fluoro-D,L-phenylalanine orgrowth of aro3 mutant yeast strains in media with excess tyrosine asdescribed by Fukuda, et al. ((1990) Breeding of Brewing Yeast Producinga Large Amount of Beta-Phenylethyl Alcohol and Beta-Phenylethyl Acetate.Agr Biol Chem Tokyo 54(1):269-271).

In certain embodiments, the engineered host cells of the presentinvention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, 13 or more, 14 or more, or even 15 or more feedbackinhibition alleviating mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 or 15 feedback inhibition alleviating mutations in one ormore biosynthetic enzyme genes within the engineered host cell.

Transcriptional Modulation Modifications

The host cells may include one or more transcriptional modulationmodifications (such as two or more, three or more, four or more, five ormore, or even more modifications) of one or more biosynthetic enzymegenes of the cell. In some examples, the one or more biosynthetic enzymegenes are native to the cell. In some examples, the one or morebiosynthetic enzyme genes are non-native to the cell. Any convenientbiosynthetic enzyme genes of the cell may be targeted for transcriptionmodulation. By transcription modulation is meant that the expression ofa gene of interest in a modified cell is modulated, e.g., increased ordecreased, enhanced or repressed, relative to a control cell (e.g., anunmodified cell). In some cases, transcriptional modulation of the geneof interest includes increasing or enhancing expression. By increasingor enhancing expression is meant that the expression level of the geneof interest is increased by 2-fold or more, such as by 5-fold or moreand sometimes by 25-, 50-, or 100-fold or more and in certainembodiments 300-fold or more or higher, as compared to a control, i.e.,expression in the same cell not modified (e.g., by using any convenientgene expression assay). Alternatively, in cases where expression of thegene of interest in a cell is so low that it is undetectable, theexpression level of the gene of interest is considered to be increasedif expression is increased to a level that is easily detectable. Incertain instances, transcriptional modulation of the gene of interestincludes decreasing or repressing expression. By decreasing orrepressing expression is meant that the expression level of the gene ofinterest is decreased by 2-fold or more, such as by 5-fold or more andsometimes by 25-, 50-, or 100-fold or more and in certain embodiments300-fold or more or higher, as compared to a control. In some cases,expression is decreased to a level that is undetectable. Modificationsof host cell processes of interest that may be adapted for use in thesubject host cells are described in U.S. Publication No. 20140273109(Ser. No. 14/211,611) by Smolke et al., the disclosure of which isherein incorporated by reference in its entirety.

Any convenient biosynthetic enzyme genes may be transcriptionallymodulated, and include but are not limited to, those biosyntheticenzymes described in FIG. 2. In particular, FIG. 2 illustrates abiosynthetic scheme for conversion of glucose to 4-HPA, dopamine, and3,4-DHPA, in accordance with embodiments of the invention. Examples ofenzymes described in FIG. 2 include ARO3, ARO4, ARO1, ARO7, TYR1, TYR,TyrH, DODC, MAO, ARO10, ARO9, and TKL. In some instances, the one ormore biosynthetic enzyme genes may be selected from ARO10, ARO9, andTKL. In some cases, the one or more biosynthetic enzyme genes may beARO10. In certain instances, the one or more biosynthetic enzyme genesmay be ARO9. In some embodiments, the one or more biosynthetic enzymegenes may be TKL. In some embodiments, the host cell includes one ormore transcriptional modulation modifications to one or more genes suchas one of those genes described in Table 2.

In some embodiments, the transcriptional modulation modification mayinclude a substitution of a strong promoter for a native promoter of theone or more biosynthetic enzyme genes or the expression of an additionalcopy(ies) of the gene or genes under the control of a strong promoter.The promoters driving expression of the genes of interest may beconstitutive promoters or inducible promoters, provided that thepromoters may be active in the host cells. The genes of interest may beexpressed from their native promoters. Additionally or alternatively,the genes of interest may be expressed from non-native promoters.Although not a requirement, such promoters may be medium to highstrength in the host in which they are used. Promoters may be regulatedor constitutive. In some embodiments, promoters that are not glucoserepressed, or repressed only mildly by the presence of glucose in theculture medium, may be used. There are numerous suitable promoters,examples of which include promoters of glycolytic genes such as thepromoter of the B. subtilis tsr gene (encoding fructose biphosphatealdolase) or GAPDH promoter from yeast S. cerevisiae (coding forglyceraldehyde-phosphate dehydrogenase) (Bitter G. A., Meth. Enzymol.152:673-684 (1987)). Other strong promoters of interest include, but arenot limited to, the ADHI promoter of baker's yeast (Ruohonen L., et al,J. Biotechnol. 39:193-203 (1995)), the phosphate-starvation inducedpromoters such as the PHO5 promoter of yeast (Hinnen A., et al, in YeastGenetic Engineering, Barr P. J., et al. eds, Butterworths (1989), thealkaline phosphatase promoter from B. licheniformis (Lee. J. W. K., etal, J. Gen. Microbiol. 137:1127-1133 (1991)), GPD1, and TEF1. Yeastpromoters of interest include, but are not limited to, induciblepromoters such as Gal1-10, Gal1, GalL, GalS, repressible promoter Met25,tetO, and constitutive promoters such as glyceraldehyde 3-phosphatedehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH),translation-elongation factor-1-alpha promoter (TEF), cytochromec-oxidase promoter (CYC1), MRP7 promoter, etc. In some instances, thestrong promoter is GPD1. In certain instances, the strong promoter isTEF1. Autonomously replicating yeast expression vectors containingpromoters inducible by hormones such as glucocorticoids, steroids, andthyroid hormones are also known and include, but are not limited to, theglucorticoid responsive element (GRE) and thyroid hormone responsiveelement (TRE), see e.g., those promoters described in U.S. Pat. No.7,045,290. Vectors containing constitutive or inducible promoters suchas alpha factor, alcohol oxidase, and PGH may be used. Additionally anypromoter/enhancer combination (as per the Eukaryotic Promoter Data BaseEPDB) could also be used to drive expression of genes of interest. It isunderstood that any convenient promoters specific to the host cell maybe selected, e.g., E. coli T7 promoter, lac promoter or tetO promoter.In some cases, promoter selection may be used to optimize transcription,and hence, enzyme levels to maximize production while minimizing energyresources.

Inactivating Mutations

The engineered host cells may include one or more inactivating mutationsto an enzyme of the cell (such as two or more, three or more, four ormore, five or more, or even more). The inclusion of one or moreinactivating mutations may modify the flux of a synthetic pathway of anengineered host cell to increase the levels of a nor-opioid ornal-opioid BIAs of interest or a desirable enzyme or precursor leadingto the same. In some examples, the one or more inactivating mutationsare to an enzyme native to the cell. Additionally or alternatively, theone or more inactivating mutations are to an enzyme non-native to thecell. As used herein, by “inactivating mutation” is meant one or moremutations to a gene or regulatory DNA sequence of the cell, where themutation(s) inactivates a biological activity of the protein expressedby that gene of interest. In some cases, the gene is native to the cell.In some instances, the gene encodes an enzyme that is inactivated and ispart of or connected to the synthetic pathway of a nor-opioid and/ornal-opioid BIAs of interest produced by the host cell. In someinstances, an inactivating mutation is located in a regulatory DNAsequence that controls a gene of interest. In certain cases, theinactivating mutation is to a promoter of a gene. Any convenientmutations (e.g., as described herein) may be utilized to inactivate agene or regulatory DNA sequence of interest. By “inactivated” or“inactivates” is meant that a biological activity of the proteinexpressed by the mutated gene is reduced by 10% or more, such as by 20%or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% ormore, 80% or more, 90% or more, 95% or more, 97% or more, or 99% ormore, relative to a control protein expressed by a non-mutated controlgene. In some cases, the protein is an enzyme and the inactivatingmutation reduces the activity of the enzyme.

In some examples, the engineered host cell includes an inactivatingmutation in an enzyme native to the cell. Any convenient enzymes may betargeted for inactivation. Enzymes of interest may include, but are notlimited to those enzymes, described in Table 2 whose action in thesynthetic pathway of the engineered host cell tends to reduce the levelsof a nor-opioid or nal-opioid of interest. In some cases, the enzyme hasglucose-6-phosphate dehydrogenase activity. In certain embodiments, theenzyme that includes an inactivating mutation is ZWF1. In some cases,the enzyme has alcohol dehydrogenase activity. In some embodiments, theenzyme that includes an inactivating mutation is selected from ADH2,ADH3, ADH4, ADH5, ADH6, ADH7, and SFA1. In certain embodiments, theenzyme that includes an inactivating mutation(s) is ADH2. In certainembodiments, the enzyme that includes an inactivating mutation(s) isADH3. In certain embodiments, the enzyme that includes an inactivatingmutation(s) is ADH4. In certain embodiments, the enzyme that includes aninactivating mutation(s) is ADH5. In certain embodiments, the enzymethat includes an inactivating mutation(s) is ADH6. In certainembodiments, the enzyme that includes an inactivating mutation(s) isADH7. In some cases, the enzyme has aldehyde oxidoreductase activity. Incertain embodiments, the enzyme that includes an inactivating mutationis selected from ALD2, ALD3, ALD4, ALD5, and ALD6. In certainembodiments, the enzyme that includes an inactivating mutation(s) isALD2. In certain embodiments, the enzyme that includes an inactivatingmutation(s) is ALD3. In certain embodiments, the enzyme that includes aninactivating mutation(s) is ALD4. In certain embodiments, the enzymethat includes an inactivating mutation(s) is ALD5. In certainembodiments, the enzyme that includes an inactivating mutation(s) isALD6. In some embodiments, the host cell includes one or moreinactivating mutations to one or more genes described in Table 2.

Epimerization Modifications

Some methods, processes, and systems provided herein describe theconversion of (S)-1-benzylisoquinoline alkaloids to(R)-1-benzylisoquinoline alkaloids. Some of these methods, processes,and systems may comprise an engineered host cell. In some examples, theconversion of (S)-1-benzylisoquinoline alkaloids to(R)-1-benzylisoquinoline alkaloids is a key step in the conversion of asubstrate to a diverse range of alkaloids. In some examples, theconversion of (S)-1-benzylisoquinoline alkaloids to(R)-1-benzylisoquinoline alkaloids comprises an epimerization reaction.In some cases, epimerization of a substrate alkaloid may be performed byoxidizing an (S)-substrate to the corresponding Schiff base or imineintermediate, then stereospecifically reducing this intermediate to an(R)-product as provided in FIG. 3 and as represented generally inScheme 1. As provided in Scheme 1, R₁, R₂, R₃, and R₄ may be H or CH₃.R₅ may be H, OH, or OCH₃.

In some examples, the conversion of the (S)-substrate to the (R)-productmay involve at least one oxidation reaction and at least one reductionreaction. In some cases, an oxidation reaction is optionally followed bya reduction reaction. In some cases, at least one of the oxidation andreduction reactions is carried out in the presence of an enzyme. In somecases, at least one of the oxidation and reduction reactions iscatalyzed by an enzyme. In some cases, the oxidation and reductionreactions are both carried out in the presence of at least one enzyme.In some cases, at least one enzyme is useful to catalyze the oxidationand reduction reactions. The oxidation and reduction reactions may becatalyzed by the same enzyme.

In some methods, processes and systems described herein, an oxidationreaction may be performed in the presence of an enzyme. In someexamples, the enzyme may be an oxidase. The oxidase may use an(S)-1-benzylisoquinoline as a substrate. The oxidase may convert the(S)-substrate to a corresponding imine or Schiff base derivative. Theoxidase may be referred to as 1,2-dehydroreticuline synthase (DRS).Non-limiting examples of enzymes suitable for oxidation of(S)-1-benzylisoquinoline alkaloids in this disclosure include acytochrome P450 oxidase, a 2-oxoglutarate-dependent oxidase, and aflavoprotein oxidase. For example, (S)-tetrahydroprotoberberine oxidase(STOX, E.C 1.3.3.8) may oxidize (S)-norreticuline and other(S)-1-benzylisoquinoline alkaloids to 1,2-dehydronorreticuline and othercorresponding 1,2-dehydro products. In some examples, a protein thatcomprises an oxidase domain of any one of the preceding examples mayperform the oxidation. In some examples, the oxidase may catalyze theoxidation reaction within a host cell, such as an engineered host cell,as described herein.

In some examples, a reduction reaction may follow the oxidationreaction. The reduction reaction may be performed by an enzyme. In someexamples, the reductase may use an imine or Schiff base derived from a1-benzylisoquinoline as a substrate. The reductase may convert the imineor Schiff base derivative to an (R)-1-benzylisoquinoline. The reductasemay be referred to as 1,2-dehydroreticuline reductase (DRR).Non-limiting examples of enzymes suitable for reduction of an imine orSchiff base derived from an (S)-1-benzylisoquinoline alkaloid include analdo-keto reductase (e.g., a codeinone reductase-like enzyme (EC1.1.1.247)) and a short chain dehydrogenase (e.g., a salutaridinereductase-like enzyme (EC 1.1.1.248)). In some examples, a protein thatcomprises a reductase domain of any one of the preceding examples mayperform the reduction. In a further embodiment, the reduction isstereospecific. In some examples, the reductase may catalyze thereduction reaction within a host cell, such as an engineered host cell,as described herein.

An example of an enzyme that can perform an epimerization reaction thatconverts (S)-1-benzylisoquinoline alkaloids to (R)-1-benzylisoquinolinealkaloids includes an epimerase having an oxidase domain and a reductasedomain. In particular, the epimerase may have a cytochrome P450 oxidase82Y2-like domain. Additionally, the epimerase may have a codeinonereductase-like domain. Further, an epimerase having a cytochrome P450oxidase 82Y2-like domain and also having a codeinone reductase-likedomain may be referred to as a CYP-COR enzyme. In particular, a CYP-CORenzyme may be a fusion enzyme. The CYP-COR enzyme may also be referredto as DRS-DRR (dehydroreticuline synthase-dehydroreticuline reductase).

An example of an amino acid sequence of a CYP-COR enzyme that may beused to perform the conversion of (S)-1-benzylisoquinoline alkaloids to(R)-1-benzylisoquinoline alkaloids is provided in FIG. 4. In particular,FIG. 4 illustrates an amino acid sequence of a CYP-COR enzyme, inaccordance with embodiments of the invention. As seen in FIG. 4,underlined text denotes the cytochrome P450 CYP82Y2-like domain (59%identity to AFB74617.1). The dotted underlined text denotes thealdo-keto reductase NADPH-dependent codeinone reductase-like domain (75%identity to ACM44066.1). Additional amino acid sequences of a CYP-CORenzyme are set forth in Table 1. An amino acid sequence for an epimerasethat is utilized in converting an (S)-1-benzylisoquinoline alkaloid toan (R)-1-benzylisoquinoline alkaloid may be 75% or more identical to agiven amino acid sequence as listed in Table 1. For example, an aminoacid sequence for such an epimerase may comprise an amino acid sequencethat is at least 75% or more, 80% or more, 81% or more, 82% or more, 83%or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% ormore, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more,94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99%or more identical to an amino acid sequence as provided herein.Additionally, in certain embodiments, an “identical” amino acid sequencecontains at least 80%-99% identity at the amino acid level to thespecific amino acid sequence. In some cases an “identical” amino acidsequence contains at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94% and more in certain cases, at least 95%, 96%, 97%, 98% and 99%identity, at the amino acid level. In some cases, the amino acidsequence may be identical but the DNA sequence is altered such as tooptimize codon usage for the host organism, for example.

An engineered host cell may be provided that produces an epimerase thatconverts (S)-1-benzylisoquinoline alkaloid to (R)-1-benzylisoquinolinealkaloid, wherein the epimerase comprises an amino acid sequenceselected from the group consisting of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, and 15. The epimerase that is produced withinthe engineered host cell may be recovered and purified so as to form abiocatalyst. In some cases, the epimerase may be split into one or moreenzymes. Additionally, one or more enzymes that are produced bysplitting the epimerase may be recovered from the engineered host cell.These one or more enzymes that result from splitting the epimerase mayalso be used to catalyze the conversion of (S)-1-benzylisoquinolinealkaloids to (R)-1-benzylisoquinoline alkaloids. In particular, the oneor more enzymes that are recovered from the engineered host cell thatproduces the epimerase may be used in a process for converting an(S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinolinealkaloid. The process may include contacting the(S)-1-benzylisoquinoline alkaloid with an epimerase in an amountsufficient to convert said (S)-1-benzylisoquinoline alkaloid to(R)-1-benzylisoquinoline alkaloid. In examples, the(S)-1-benzylisoquinoline alkaloid may be contacted with a sufficientamount of the one or more enzymes such that at least 5% of said(S)-1-benzylisoquinoline alkaloid is converted to(R)-1-benzylisoquinoline alkaloid. In further examples, the(S)-1-benzylisoquinoline alkaloid may be contacted with a sufficientamount of the one or more enzymes such that at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 82%, at least 84%, atleast 86%, at least 88%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, at least 99.7%, or 100% of said(S)-1-benzylisoquinoline alkaloid is converted to(R)-1-benzylisoquinoline alkaloid.

The one or more enzymes that may be used to convert an(S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinolinealkaloid may contact the (S)-1-benzylisoquinoline alkaloid in vitro.Additionally, or alternatively, the one or more enzymes that may be usedto convert an (S)-1-benzylisoquinoline alkaloid to an(R)-1-benzylisoquinoline alkaloid may contact the(S)-1-benzylisoquinoline alkaloid in vivo. Additionally, the one or moreenzymes that may be used to convert an (S)-1-benzylisoquinoline alkaloidto an (R)-1-benzylisoquinoline alkaloid may be provided to a cell havingthe (S)-1-benzylisoquinoline alkaloid within, or may be produced withinan engineered host cell.

In some examples, the methods provide for engineered host cells thatproduce an alkaloid product, wherein the epimerization of an(S)-substrate to an (R)-product may comprise a key step in theproduction of an alkaloid product. In some examples, the alkaloidproduced is an (R)-1-benzylisoquinoline alkaloid. In still otherembodiments, the alkaloid produced is derived from an(R)-1-benzylisoquinoline alkaloid, including, for example, 4-ringpromorphinan and 5-ring morphinan alkaloids. In another embodiment, an(S)-1-benzylisoquinoline alkaloid is an intermediate toward the productof the engineered host cell. In still other embodiments, the alkaloidproduct is selected from the group consisting of morphinan andpromorphinanalkaloids.

In some examples, the (S)-substrate is an (S)-1-benzylisoquinolinealkaloid selected from the group consisting of (S)-norreticuline,(S)-reticuline, (S)-tetrahydropapaverine, (S)-norcoclaurine,(S)-coclaurine, (S)—N-methylcoclaurine,(S)-3′-hydroxy-N-methylcoclaurine, (S)-norisoorientaline,(S)-orientaline, (S)-isoorientaline, (S)-norprotosinomenine,(S)-protosinomenine, (S)-norlaudanosoline, (S)-laudanosoline,(S)-4′-O-methyllaudanosoline, (S)-6-O-methylnorlaudanosoline,(S)-4′-O-methylnorlaudanosoline.

In some examples, the (S)-substrate is a compound of Formula I:

or a salt thereof, wherein:

R¹, R², R³, and R⁴ are independently selected from hydrogen and methyl;and

R⁵ is selected from hydrogen, hydroxy, and methoxy.

In some other examples, at least one of R¹, R², R³, R⁴, and R⁵ ishydrogen.

In still other examples, the (S)-substrate is a compound of Formula II:

or a salt thereof, wherein:

R³ is selected from hydrogen and C₁-C₄ alkyl;

R⁶ and R⁷ are independently selected at each occurrence from hydroxy,fluoro, chloro, bromo, carboxaldehyde, C₁-C₄ acyl, C₁-C₄ alkyl, andC₁-C₄ alkoxy;

n is 0, 1, 2, 3, or 4; and

n′ is 0, 1, 2, 3, 4 or 5.

When a bond is drawn across a ring, it means substitution may occur at anon-specific ring atom or position. For example, in Formula II shownabove, the hydrogen of any —CH— in the 6-membered ring may be replacedwith R⁷ to form —CR⁷—.

In some examples, R⁶ and R⁷ are independently methyl or methoxy. In someother examples, n and n′ are independently 1 or 2. In still otherembodiments, R³ is hydrogen or methyl.

In some examples, the methods provide for engineered host cells thatproduce alkaloid products from (S)-reticuline. The epimerization of(S)-reticuline to (R)-reticuline may comprise a key step in theproduction of diverse alkaloid products from a precursor. In someexamples, the precursor is L-tyrosine or a sugar (e.g., glucose). Thediverse alkaloid products can include, without limitation, morphinan andpromorphinan alkaloids.

Any suitable carbon source may be used as a precursor toward anepimerized 1-benzylisoquinoline alkaloid. Suitable precursors caninclude, without limitation, monosaccharides (e.g., glucose, fructose,galactose, xylose), oligosaccharides (e.g., lactose, sucrose,raffinose), polysaccharides (e.g., starch, cellulose), or a combinationthereof. In some examples, unpurified mixtures from renewable feedstockscan be used (e.g., corn steep liquor, sugar beet molasses, barley malt,biomass hydrolysate). In still other embodiments, the carbon precursorcan be a one-carbon compound (e.g., methanol, carbon dioxide) or atwo-carbon compound (e.g., ethanol). In yet other embodiments, othercarbon-containing compounds can be utilized, for example, methylamine,glucosamine, and amino acids (e.g., L-tyrosine). In some examples, a1-benzylisoquinoline alkaloid may be added directly to an engineeredhost cell, including, for example, norlaudanosoline, laudanosoline,norreticuline, and reticuline. In still further embodiments, a1-benzylisoquinoline alkaloid may be added to the engineered host cellas a single enantiomer (e.g., an (S)-1-benzylisoquinoline alkaloid), ora mixture of enantiomers, including, for example, a racemic mixture.

In some examples, the methods provide for the epimerization of astereocenter of a 1-benzylisoquinoline alkaloid, or a derivativethereof. In a further embodiment, the method comprises contacting the1-benzylisoquinoline alkaloid with at least one enzyme. The at least oneenzyme may invert the stereochemistry of a stereocenter of a1-benzylisoquinoline alkaloid, or derivative thereof, to the oppositestereochemistry. In some examples, the at least one enzyme converts an(S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinolinealkaloid. In some examples of this conversion of an(S)-1-benzylisoquinoline alkaloid to an (R)-1-benzylisoquinolinealkaloid utilizing the at least one enzyme, the (S)-1-benzylisoquinolinealkaloid is selected from the group consisting of (S)-norreticuline,(S)-reticuline, (S)-tetrahydropapaverine, (S)-norcoclaurine,(S)-coclaurine, (S)—N-methylcoclaurine,(S)-3′-hydroxy-N-methylcoclaurine, (S)-norisoorientaline,(S)-orientaline, (S)-isoorientaline, (S)-norprotosinomenine,(S)-protosinomenine, (S)-norlaudanosoline, (S)-laudanosoline,(S)-4′-O-methyllaudanosoline, (S)-6-O-methylnorlaudanosoline, and(S)-4′-O-methylnorlaudanosoline.

In still other embodiments, the 1-benzylisoquinoline alkaloid that isepimerized may comprise two or more stereocenters, wherein only one ofthe two or more stereocenters is inverted to produce a diastereomer ofthe substrate (e.g., (S, R)-1-benzylisoquinoline alkaloid converted to(R, R)-1-benzylisoquinoline alkaloid). In examples where only onestereocenter of a 1-benzylisoquinoline alkaloid is inverted whencontacted with the at least one enzyme, the product is referred to as anepimer of the 1-benzylisoquinoline alkaloid.

In some examples, the 1-benzylisoquinoline alkaloid is presented to theenzyme as a single stereoisomer. In some other examples, the1-benzylisoquinoline alkaloid is presented to the enzyme as a mixture ofstereoisomers. In still further embodiments, the mixture ofstereoisomers may be a racemic mixture. In some other examples, themixture of stereoisomers may be enriched in one stereoisomer as comparedto another stereoisomer.

In some examples, an 1-benzylisoquinoline alkaloid, or a derivativethereof, is recovered. In some examples, the 1-benzylisoquinolinealkaloid is recovered from a cell culture. In still further embodiments,the recovered 1-benzylisoquinoline alkaloid is enantiomerically enrichedin one stereoisomer as compared to the original mixture of1-benzylisoquinoline alkaloids presented to the enzyme. In still furtherembodiments, the recovered 1-benzylisoquinoline alkaloid has anenantiomeric excess of at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 82%, atleast 84%, at least 86%, at least 88%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%,or 100%.

“Isomers” are different compounds that have the same molecular formula.“Stereoisomers” are isomers that differ only in the way the atoms arearranged in space. “Enantiomers” are a pair of stereoisomers that arenon-superimposable mirror images of each other. A 1:1 mixture of a pairof enantiomers is a “racemic” mixture. “Diastereoisomers” or“diastereomers” are stereoisomers that have at least two asymmetricatoms but are not mirror images of each other. The term “epimer” as usedherein refers to a compound having the identical chemical formula but adifferent optical configuration at a particular position. For example,the (R,S) and (S,S) stereoisomers of a compound are epimers of oneanother. In some examples, a 1-benzylisoquinoline alkaloid is convertedto its epimer (e.g., epi-1-benzylisoquinoline alkaloid). The absolutestereochemistry is specified according to the Cahn-Ingold-Prelog R-Ssystem. When a compound is a pure enantiomer, the stereochemistry ateach chiral carbon can be specified by either R or S. Resolved compoundswhose absolute configuration is unknown can be designated (+) or (−)depending on the direction (dextro- or levorotatory) in which theyrotate plane polarized light at the wavelength of the sodium D line.Certain compounds described herein contain one or more asymmetriccenters and can thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that can be defined, in terms of absolutestereochemistry, as (R)- or (S)—.

TABLE 1  Example partial and full-length amino acid sequences ofCYP-COR fusion enzymes. SEQ ID Sequence Description NO.MELQYISYFQPTSSVVALLLALVSILSSVVVLRKTFLNNY P. somniferum SEQ. IDSSSPASSTKTAVLSHQRQQSCALPISGLLHIFMNKNGLIH plant source; NO. 1VTLGNMADKYGPIFSFPTGSHRTLVVSSWEMVKECFTGNN full-lengthDTAFSNRPIPLAFKTIFYACGGIDSYGLSSVPYGKYWREL amino acidRKVCVHNLLSNQQLLKFRHLIISQVDTSFNKLYELCKNSE sequenceDNHGNYTTTTTTAAGMVRIDDWLAELSFNVIGRIVCGFQS >RQNK-GPKTGAPSRVEQFKEAINEASYFMSTSPVSDNVPMLGWID 2062398QLTGLTRNMKHCGKKLDLVVESIINDHRQKRRFSRTKGGD (also FPYZ-EKDDEQDDFIDICLSIMEQPQLPGNNNPSQIPIKSIVLDM 2037562,IGGGTDTTKLTTIWTLSLLLNNPHVLDKAKQEVDAHFRTK BMRX-RRSTNDAAAAVVDFDDIRNLVYIQAIIKESMRLYPASPVV 2007040, andERLSGEDCVVGGFHVPAGTRLWANVWKMQRDPKVWDDPLV MLPX-FRPDRFLSDEQKMVDVRGQNYELLPFGAGRRVCPGVSFSL 2016197)DLMQLVLTRLILEFEMKSPSGKVDMTATPGLMSYKVIPLDILLTHRRIKPCVQSAASERDMESSGVPVITLGSGKVMPVLGMGTFEKVGKGSERERLAILKAIEVGYRYFDTAAAYETEEVLGEAIAEALQLGLVKSRDELFISSMLWCTDAHADRVLLALQNSLRNLKLEYVDLYMLPFPASLKPGKITMDIPEEDICRMDYRSVWAAMEECQNLGFTKSIGVSNFSCKKLQELMATANIPPAVNQVEMSPAFQQKKLREYCNANNILVSAISVLGSNGTPWGSNAVLGSEVLKKIAMAKGKSVAQVSMRWVYEQGASLVVKSFSEERLRENLNIFDWELTKEDHEKIGEIPQCRILSA YFLVSPNGPFKSQEELWDDEA*MELQYISYFQPTSSVVALLLALVSILSSVVVLRKTFLNNY P. somniferum SEQ. IDSSSPASSTKTAVLSHQRQQSCALPISGLLHIFMNKNGLIH plant source; NO. 2VTLGNMADKYGPIFSFPTGSHRTLVVSSWEMVKECFTGNN full-lengthDTAFSNRPIPLAFKTIFYACGGIDSYGLSSVPYGKYWREL amino acidRKVCVHNLLSNQQLLKFRHLUSQVDTSFNKLYELCKNSED sequenceNHGNYTTXLLLPQLAWRQPWKLYYXTTTTAAGMVRIDDWL >KKCW-AELSFNVIGRIVCGFQSGPKTGAPSRVEQFKEAINEASYF 2026866MSTSPVSDNVPMLGWIDQLTGLTRNMKHCGKKLDLVVESU (also FPYZ-NDHRQKRRFSRTKGGDEKDDEQDDFIDICLSIMEQPQLPG 2037562,NNNPSQIPIKSIVLDMIGGGTDTTKLTTrWTLSLLLNNPH MLPX-VLDKAKQEVDAHFRTKRRSTNDAAAAVVDFDDIRNLVYIQ 2016197)AIIKESMRLYPASPVVERLSGEDCVVGGFHVPAGTRLWANVWKMQRDPKVWDDPLVFRPDRFLSDEQKMVDVRGQNYELLPFGAGRRVCPGVSFSLDLMQLVLTRLILEFEMKSPSGKVDMTATPGLMSYKVIPLDILLTHRRIRPCVQSAASERDMESSGVPVITLGSGKVMPVLGMGTFEKVGKGSERERLAILKAIEVGYRYFDTAAAYETEEVLGEAIAEALQLGLVKSRDELFISSMLWCTDAHADRVLLALQNSLRNLKLEYVDLYMLPFPASLKPGKITMDIPEEDICRMDYRSVWAAMEECQNLGFTKSIGVSNFSCKKLQELMATANIPPAVNQVEMSPAFQQKKLREYCNANNILVSAISVLGSNGTPWGSNAVLGSEVLKKIAMAKGKSVAQVSMRWVYEQGASLVVKSFSEERLRENLNIFDWELTKEDHEKIGEIPQCRILSAYFLVSPNGPFKSQEELWDDEA*MELQYISYFQPTSSVVALLLALVSILSSVVVLRKTFLNNYS P. somniferum SEQ. IDSSPASSTKTAVLSHQRQQSCALPISGLLHIFMNKNGLIHVT plant source; NO. 3LGNMADKYGPIFSFPTGSHRTLVVSSWEMVKECFTGNNDTA partial-lengthFSNRPIPLAFKTIFYACGGIDSYGLSSVPYGKYWRELRKVC amino acidVHNLLSNQQLLKFRHLIISQVDTSFNKLYELCKNSEDNHGN sequenceYTTTTTTAAGMVRIDDWLAELSFNVIGRIVCGFQSGPKTGA >SUFP-PSRVEQFKEAINEASYFMSTSPVSDNVPMLGWIDQLTGLTR 2025636NMKHCGKKLDLVVESIINDHRQKRRFSRTKGGDEKDDEQDDFIDICLSIMEQPQLPGNNNPSQIPIKSIVLDMIGGGTDTTKLTTIWTLSLLLNNPHVLDKAKQEVDAHFRTKRRSTNDAAAAVVDFDDIRNLVYIQAIIKESMRLYPASPVVERLSGEDCVVGGFHVPAGTRLWANVWKMQRDPKVWDDPLVFRPDRFLSDEQKMVDVRGQNYELLPFGAGRRVCPGVSFSLDLMQLVLTRLILEFEMKSPSGKVDMTATPGLMSYKVIPLDILLTHRRIRPCVQSAASERDMESSGVPVITLGSGKVMPVLGMGTFEKVGKGSERERLAILKAIEVGYRYFDTAAAYETEEVLGEAIAEALQLGLVKSRDELFISSMLWCTDAHADRVLLALQNSLRNLKLEYVDLYMLPFPASLKPGKITMDIPEEDICRMDYRXVSKPWLH*MRWHRXIDSYGLSSVPYGKYWRELRKVCVHNLLSNQQLLK P. somniferum SEQ. IDFRHLIISQVDTSFNKLYELCKNSEDNQGNYPTTTTAAGMVR plant source; NO. 4IDDWLAELSFNVIGRIVCGFQSGPKTGAPSRVEQFKEAINE partial-lengthASYFMSTSPVSDNVPMLGWIDQLTGLTRNMKHCGKKLDLVV amino acidESIINDHRQKRRFSRTKGGDEKDDEQDDFIDICLSIMEQPQ sequenceLPGNNNPSQIPIKSIVLDMIGGGTDTTKLTTIWTLSLLLNN >MIKW-PHVLDKAKQEVDAHFRTKRRSTNDAAAAVVDFDDIRNLVYI 2013651QAIIKESMRLYPASPVVERLSGEDCVVGGFHVPAGTRLWANVWKMQRDPKVWDDPLVFRPDRFLSDEQKMVDVRGQNYELLPFGAGRRVCPGVSFSLDLMQLVLTRLILEFEMKSPSGKVDMTATPGLMSYKVIPLDILLTHRRIKPCVQSAASERDMESSGVPVITLGSGKVMPVLGMGTFEKVGKGSERERLAILKAIEVGYRYFDTAAAYETEEVLGEAIAEALQLGLVKSRDELFISSMLWCTDAHADRVLLALQNSLRNLKLEYVDLYMLPFPASLKPGKITMDIPEEDICRMDYRSVWAAMEECQNLGFTKSIGVSNFSCKKLQELMATANIPPAVNQVEMSPAFQQKKLREYCNANNILVSAISVLGSNGTPWGSNAVLGSEVLKKIAMAKGKSVAQVSMRWVYEQGASLVVKSFSEERLRENLNIFDWELTKEDHEKIGEIPQCRILSAYFLVSPNGPFKSQEELWDDEA*MELQYISYFQPTSSVVALLLALVSILSSVVVLRKTFLNNYS P. setigerum SEQ. IDSSPASSTKTAVLSHQRQQSCALPISGLLHIFMNKNGLIHVT plant source; NO. 5LGNMADKYGPIFSFPTGSHRTLVVSSWEMVKECFTGNNDTA full-lengthFSNRPIPLAFKTIFYACGGIDSYGLSSVPYGKYWRELRKVC amino acidVHNLLSNQQLLKFRHLIISQVDTSFNKLYELCKNSEDNQGN sequenceYTTTTTAAGMVRIDDWLAELSFNVIGRIVCGFQSGPKTGAP >EPRK-SRVEQFKEAINEASYFMSTSPVSDNVPMLGWIDQLTGLTRN 2027940MKFICGKKLDLVVESIINDHRQKRRFSRTKGGDEKDDEQDD (also FPYZ-FIDICLSIMEQPQLPGNNNPSQIPIKSIVLDMIGGGTDTTK 2037562,LTTIWTLSLLLNNPHVLDKAKQEVDAHFRTKRRSTNDAAAA STDO-VVDFDDIRNLVYIQAIIKESMRLYPASPVVERLSGEDCVVG 2019715,GFHVPAGTRLWANVWKMQRDPKVWDDPLVFRPDRFLSDEQK FNXH-MVDVRGQNYELLPFGAGRRVCPGVSFSLDLMQLVLTRLIL 2029312,EFEMKSPSGKVDMTATPGLMSYKVIPLDILLTHRRIKPCVQ MLPX-SAASERDMESSGVPVITLGSGKVMPVLGMGTFEKVGKGSE 2016196,RERLAILKAIEVGYRYFDTAAAYETEEVLGEAIAEALQLGL MLPX-VKSRDELFISSMLWCTDAHADRVLLALQNSLRNLKLEYVD 2016197)LYMLPFPASLKPGKITMDIPEEDICRMDYRSVWAAMEECQNLGFTKSIGVSNFSCKKLQELMATANIPPAVNQVEMSPAFQQKKLREYCNANNILVSAISVLGSNGTPWGSNAVLGSEVLKKIAMAKGKSVAQVSMRWVYEQGASLVVKSFSEERLRENLNIFDWELTKEDHEKIGEIPQCRILSAYFLVSPNGPFKSQEELW DDEA*MELQYISYFQPTSSVVALLLALVSILSSVVVLRKTFLNNYS P. setigerum SEQ. IDSSPASSTKTAVLSFIQRQQSCALPISGLLHIFMNKNGLIHV plant source; NO. 6TLGNMADKYGPIFSFPTGSHRTLVVSSWEMVKECFTGNNDT partial-lengthAFSNRPIPLAFKTIFYACGGIDSYGLSSVPYGKYWRELRKV amino acidCVHNLLSNQQLLKFRHLIISQVDTSFNKLYELCKNSEDNQG sequenceNYTTTTTAAGMVRIDDWLAELSFNVIGRIVCGFQSGPKTGA >QCOU-PSRVEQFKEAINEASYFMSTSPVSDNVPMLGWIDQLTGLTR 2000833NMKFICGKKLDLVVESIINDHRQKRRFSRTKGGDEKDDEQDDFIDICLSIMEQPQLPGNNNPSQIPIKSIVLDMIGGGTDTTKLTTIWTLSLLLNNPHVLDKAKQEVDAHFRTKRRSTNDAAAAVVDFDDIRNLVYIQALYPASPVVERLSGEDCVVGGFHVPAGTRLWANVWKMQRDPKVWDDPLVFRPDRFLSDEQKMVDVRGQNYELLPFGAGRRVCPGVSFSLDLMQLVLTRLILEFEMKSPSGKVDMTATPGLMSYKVIPLDILLTHRRIKPCVQSAASERDMESSGVPVITLGSGKVMPVLGMGTFEKVGKGSERERLAILKAIEVGYRYFDTAAAYETEEVLGEAIAEALQLGLVKSRDELFISSMLWCTDAHADRVLLALQNSLRNLKLEYVDLYMLPFP ASLKPGKITMDIPEEDICRMDYRSVWAAMEEMELQYFSYFQPTSSVVALLLALVSILFSVVVLRKTFSNNYS P. bracteatum SEQ. IDSPASSTETAVLCHQRQQSCALPISGLLHVFMNKNGLIHVTL plant source; NO. 7GNMADKYGPIFSFPTGSHRTLVVSSWEMVKECFTGNNDTAF full-lengthSNRPIPLAFQTIFYACGGIDSYGLSSVPYGKYWRELRKVCV amino acidHNLLSNQQLLKFRHLIISQVDTSFNKLYELCKNSEDNQGMV sequenceRMDDWLAQLSFNVIGRIVCGFQSDPKTGAPSRVEQFKEVIN >SSDU-EASYFMSTSPVSDNVPMLGWIDQLTGLTRNMKHCGKJCLD 2015634LVVESIIKDHRQKRRFSRTKGGDEKDDEQDDFIDICLSIME (also SSDU-QPQLPGNNSPPQIPIKSIVLDMIGGGTDTTKLTTIWTLSLL 2015636,LNNPHVLDKAKQEVDAHFRKKRRSTDDAAAAVVDFDDIRNL ZSNV-VYIQAIIKESMRLYPASPVVERLSGEDCVVGGFHVPAGTRL 2027701,WANVWKMQRDPKVWDDPLVFRPERFLSDEQKMVDVRGQNY RRID-ELLPFGAGRRICPGVSFSLDLMQLVLTRLILEFEMKSPSGK 2004435)VDMTATPGLMSYKVVPLDILLTHRRIKSCVQLASSERDMESSGVPVITLSSGKVMPVLGMGTFEKVGKGSERERLAILKAIEVGYRYFDTAAAYETEEVLGEAIAEALQLGLIESRDELFISSMLWCTDAHPDRVLLALQNSLRNLKLEYLDLYMLPFPASLKPGKITMDIPEEDICRMDYRSVWSAMEECQNLGFTKSIGVSNFSSKKLQELMATANIPPAVNQVEMSPAFQQKKLREYCNANNILVSAVSILGSNGTPWGSNAVLGSEVLKQIAMAKGKSVAQVSMRWVYEQGASLVVKSFSEERLRENLNIFDWELTKEDNEKIGEIPQCRILTAYFLVSPNGPFKSQEELWDDKA*MELQYFSYFQPTSSVVALLLALVSILFSVVVLRKTFSNNYS P. bracteatum SEQ. IDSPASSTETAVLCHQRQQSCALPISGLLHVFMNKNGLIHVTL plant source; NO. 8GNMADKYGPIFSFPTGSHRTLVVSSWEMVKECFTGNNDTAF full-lengthSNRPIPLAFQTIFYACGGIDSYGLSSVPYGKYWRELRKVCV amino acidHNLLSNQQLLKFRHLIISQVDTSFNKLYELCKNSEDNQGMV sequenceRMDDWLAQLSFNVIGRIVCGFQSDPKTGAPSRVEQFKEVIN >TMWO-EASYFMSTSPVSDNVPMLGWIDQLTGLTRNMKHCGKKLDL 2027322VVESIIKDHRQKRRFSRTKGGDEKDDEQDDFIDICLSIMEQ (also RRID-PQLPGNNSPPQIPIKSIVLDMIGGGTDTTKLTTIWTLSLLL 2004435)NNPHVLDKAKQEVDAHFRKKRRSTDDAAAAVVDFDDIRNLVYIQAIIKESMRLYPASPVVERLSGEDCVVGGFHVPAGTRLWANVWKMQRDPKVWDDPLVFRPERFLSDEQKMVDVRGQNYELLPFGAGRRICPGVSFSLDLMQLVLTRLILEFEMKSPSGKVDMTATPGLMSYKVVPLDILLTHRRIKSCVQLASSERDMESSGVPVITLSSGKVMPVLGMGTFEKVGKGSERERLAILKAIEVGYRYFDTAAAYETEEVLGEAIAEALQLGLIESRDELFISSMLWCTDAHPDRVLLALQNSLRNLKLEYLDLYMLPFPASLKPGKITMDIPEEDICRMDYRSVWSAMEECQNLGFTKSIGVSNFSCKKLQELMATANIPPAVNQVEMSPAFQQKKLREYCNANNILVSAVSILGSNGTPWGSNAVLGSEVLKQIAMAKGKSVAQVSMRWVYEQGASLVVKSFSEERLRENLNIFDWELTKEDNEKIGEIPQCRILTAYFLVSPNGPFKSQEELWDDKA*SSPASSTETAVLCHQRQQSCALPISGLLHIFMNKNGLIHVT P. bracteatum SEQ. IDLGNMADKYGPIFSFPTGSIIRILVVSSWEMVKECFTGNNDT plant source; NO. 9AFSNRPIPLAFKTIFYACRGIDSYGLSSVPYGKYWRELRKV partial-lengthCVHNLLSNQQLLKFRHLIISQVDTSFNKLYELCKNSEDNQG amino acidMVRMDDWLAQLSFSVIGRIVCGFQSDPKTGAPSRVEQFKEA sequenceINEASYFMSTSPVSDNVPMLGWID0LTGLTRNMTHCGKKLD >pbr.PBRST1LVVESIINDHRQKRRFSRTKGGDEKDDEQDDFIDICLSIME PF_89405QPQLPGNNNPPKIPIKSIVLDMIGAGTDTTKLTIIWTLSLLLNNPNVLAKAKQEVDAHFETKKRSTNEASVVVDFDDIGNLVYIQAIIKESMRLYPVSPVVERLSSEDCVVGGFHVPAGTRLWANVWKMQRDPKVWDDPLVFRPERFLSDEQKMVDVRGQNYELLPFGAGRRICPGVSFSLDLMQLVLTRLILEFEMKSPSGKVDMTATPGLMSYKVVPLDILLTHRRIKSCVQLASSERDMESSGVPVITLRSGKVMPVLGMGTFEKAGKGSERERLAILKAIEVGYRYFDTAAAYETEEVLGEAIAEALQLGLIKSRDELFISSMLWCTDAHPDRVLLALQNSLRNLKLEYVDLYMLPFPASLKPGKITMDIPEEDICPMDYRSVWSAMEECQNLGLTKSIGVSNFSCKKLEELMATANIPPAVNQVEMSPAFQQKKLREYCNANNILVSAVSILGSNGTPWGSNAVLGSEVLKKIAMAKGKSVAQVSMRWVYEQGASLVVKSFSEERLRENLNIFDWQLTKEDNEKIGEIPQCRILSAYFLVSPKGPFKSQEELWDDKA*SSPASSTETAVLCHQRQQSCALPISGLLHIFMNKNGLIHVT P. bracteatum SEQ. IDLGNMADKYGPIFSFPTGSHRILVVSSWEMVKECFTGNNDTF plant source; NO. 10FSNRPIPLAFKIIFYAGGVDSYGLALVPYGKYWRELRKICV partial-lengthHNLLSNQQLLKFRHLIISQVDTSFNKLYELCKNSEDNQGMV amino acidRMDDWLAQLSFSVIGRIVCGFQSDPKTGAPSRVEQFKEAIN sequenceEASYFMSTSPVSDNVPMLGWIDQLTGLTRNMTHCGKKLDLV >pbr.PBRST1VESIINDHRQKRRFSRTKGGDEKDDEQDDFIDICLSIMEQP PF_4328QLPGNNNPPKIPIKSIVLDMIGGGTDTTKLTTIWTLSLLLNNPHVLDKAKQEVDAHFLTKRRSTNDAAVVDFDDIRNLVYIQAIIKESMRLYPASPVVERLSGEDCVVGGFHVPAGTRLWVNVWKMQRDPNVWADPMVFRPERFLSHGQKKMVDVRGKNYELLPFGAGRRICPGISFSLDLMQLVLTRLILEFEMKSPSGKVDMTATPGLMSYKVVPLDILLTHRRIKSCVQLASSERDMESSGVPVITLRSGKVMPVLGMGTFEKAGKGSERERLAILKAIEVGYRYFDTAAAYETEEVLGEAIAEALQLGLIKSRDELFISSMLWCTDAHPDRVLLALQNSLRNLKLEYVDLYMLPFPASLKPGKJTMDIPEEDICPMDYRSVWSAMEECQNLGLTKSIGVSNFSCKKLEELMATANIPPAVNQVEMSPAFQQKKLREYCNANNILVSAVSILGSNGTPWGSNAVLGSEVLKKIAMAKGKSVAQVSMRWVYEQGASLVVKSFSEERLRENLNIFDWQLTKEDNEKIGEIPQCRILSAYFLVSPKGPFKSQEELWDDKA*SSPASSTETAVLCHQRQQSCALPISGLLHIFMNKNGLIHVT P. bracteatum SEQ. IDLGNMADKYGPIFSFPTGSHRILVVSSWEMVKECFTGNNDTF plant source; NO. 11FSNRPIPLAFKIIFYAGGVDSYGLALVPYGKYWRELRKICV partial-lengthHNLLSNQQLLNFRHLIISQVDTSFNKLYDLSNKKKNTTTDS amino acidGTVRMDDWLAQLSFNVIGRIVCGFQTHTETSATSSVERFTE sequenceAIDEASRFMSIATVSDTFPWLGWIDQLTGLTRKMKHYGKKL >pbr.PBRST1DLVVESIIEDHRQNRRISGTKQGDDFIDICLSIMEQPQIIP PF_12180GNNDPPRQIPIKSIVLDMIGGGTDTTKLTTTWTLSLLLNNPHVLEKAREEVDAHFGTKRRPTNDDAVMVEFDDIRNLVYIQAIIKESMRLYPASPVVERLSGEDCVVGGFHVPAGTRLWVNVWKMQRDPNVWADPMVFRPERFLSDEQKMVDVRGQNYELLPFGAGRRICPGVSFSLDLMQLVLTRLILEFEMKSPSGKVDMTATPGLMSYKVVPLDILLTHRRIKSCVQLASSERDMESSGVPVITLRSGKVMPVLGMGTFEKAGKGSERERLAILKAIEVGYRYFDTAAAYETEEVLGEAIAEALQLGLIKSRDELFISSMLWCTDAHPDRVLLALQNSLRNLKLEYVDLYMLPFPASLKPGKITMDIPEEDICPMDYRSVWSAMEECQNLGLTKSIGVSNFSCKKLEELMATANIPPAVNQVEMSPAFQQKKLREYCNANNILVSAVSILGSNGTPWGSNAVLGSEVLKKIAMAKGKSVAQVSMRWVYEQGASLVVKSFSEERLRENLNIFDWQLTKEDNEKIGEI PQCRILSAYFLVSPKGPFKSQEELWDDKA*VALRKKILKNYYSSSSSTATAVSHQWPKASRALPLIDLLHV P. bracteatum SEQ. IDFFNKTDLMHVTLGNMADKFGPIFSFPTGSHRTLVVSSWEKA plant source; NO. 12KECFTGNNDIVFSGRPLPLAFKLIFYAGGIDSYGISQVPYG partial-lengthKKWRELRNICVHNILSNQQLLKFRHLMISQVDNSFNKLYEV amino acidCNSNKDEGDSATSTTAAGIVRMDDWLGKLAFDVIARIVCG sequenceFQSQTETSTTSSMERFTEAMDEASRFMSVTAVSDTVPWLG >pbr.PBRST1WIDQLTGLKRNMKHCGKKLNLVVKSIIEDHRQKRRLSSTK PF_4329KGDENIIDEDEQDDFIDICLSIMEQPQLPGNNNPPKIPIKSIVLDMIGGGTDTTKLTTIWTLSLLLNNPHVLDKAKQEVDAHFLTKRRSTNDAAVVDFDDIRNLVYIQAIIKESMRLYPASPVVERLSGEDCVVGGFHVPAGTRLWVNVWKMQRDPNVWADPMVFRPERFLSDEQKMVDVRGQNYELLPFGAGRRICPGVSFSLDLMQLVLTRLILEFEMKSPSGKVDMTATPGLMSYKVVPLDILLTHRRIKSCVQLASSERDMESSGVPVITLRSGKVMPVLGMGTFEKAGKGSERERLAILKAIEVGYRYFDTAAAYETEEVLGEAIAEALQLGLIKSRDELFISSMLWCTDAHPDRVLLALQNSLRNLKLEYVDLYMLPFPASLKPGKITMDIPEEDICPMDYRSVWSAMEECQNLGLTKSIGVSNFSCKKLEELMATANIPPAVNQVEMSPAFQQKKLREYCNANNILVSAVSILGSNGTPWGSNAVLGSEVLKKIAMAKGKSVAQVSMRWVYEQGASLVVKSFSEERLRENLNIFDWQLTKEDNEKJGEIPQCRILSAYFLVS PKGPFKSQEELWDDKA*MELQYFSYFQPTSSVVALLLALVSILFSVVVLRKTFSNNY P. bracteatum SEQ. IDSSPASSTETAVLCHQRQQSCALPISGLLHVFMNKNGLIHV plant source; NO. 13TLGNMADKYGPIFSFPTGSHRTLVVSSWEMVKECFTGNND partial-lengthTAFSNRPIPLAFQTIFYACGGIDSYGLSSVPYGKYWRELR amino acidKVCVHNLLSNQQLLKFRHLIISQVDTSFNKLYELCKNSED sequenceNQGMVRMDDWLAQLSFNVIGRIVCGFQSDPKTGAPSRVEQ >SSDU-FKEVINEASYFMSTSPVSDNVPMLGWIDQLTGLTRNMKHC 2015635GKKLDLVVESIIKDHRQKRRFSRTKGGDEKDDEQDDFIDICLSIMEQPQLPGNNSPPQIPIKSIVLDMIGGGTDTTKLTTIWTLSLLLNNPHVLDKAKQEVDAHFRKKRRSTDDAAAAVVDFDDIRNLVYIQAIIKESMRLYPASPVVERLSGEDCVVGGFHVPAGTRLWANVWKMQRDPKVWDDPLVFRPERFLSDEQKMVDVRGQNYELLPFGAGRRICPGVSFSLDLMQLVLTRLILEFEMKSPSGKVDMTATPGLMSYKVVPLDILLTHRRIKSCVQLASSERDMESSGVPVITLSSGKVMPVLGMGTFEKVGKGSERERLAILKAIEVGYRYFDTAAAYETEEVLGEAIAEALQLGLIESRDELFISSMLWCTDAHPDRYLLALQNSLRNLKLEYLDLYMLPFPASLKPGKITMDIPEEDICRMDYRSVWSAMEECQNLGFTKSIGVSNFSSKKLQELMATANIPPAVNQVEMSPAFQQKKLREYCNANNILVSAVSILGSNGTPWGSNAVLGSEVLKQIAMAKGKSVAQVSMRWVXKFSAYAIVWSLFFGHRIC ITLYSFLIRNVAYICITY*MELQYFSYFQPTSSVVALLLALVSILFSVVVLRKTFSNNY P. bracteatum SEQ. IDSSPASSTETAVLCHQRQQSCALPISGLLHVFMNKNGLIHV plant source; NO. 14TLGNMADKYGPIFSFPTGSHRTLVVSSWEMVKECFTGNND partial-lengthTAFSNRPIPLAFQTIFYACGGIDSYGLSSVPYGKYWRELR amino acidKVCVHNLLSNQQLLKFRHLIISQVDTSFNKLYELCKNSED sequenceNQGMVRMDDWLAQLSFNVIGRIVCGFQSDPKTGAPSRVEQ >SSDU-FKEVINEASYFMSTSPVSDNVPMLGWIDQLTGLTRNMKHC 2015637GKKLDLVVESIIKDHRQKRRFSRTKGGDEKDDEQDDFIDICLSIMEQPQLPGNNSPPQIPIKSIVLDMIGGGTDTTKLTTIWTLSLLLNNPHVLDKAKQEVDAHFRKKRRSTDDAAAAVVDFDDIRNLVYIQAIIKESMRLYPASPVVERLSGEDCVVGGFHVPAGTRLWANVWKMQRDPKVWDDPLVFRPERFLSDEQKMVDVRGQNYELLPFGAGRRICPGVSFSLDLMQLVLTRLILEFEMKSPSGKVDMTATPGLMSYKVVPLDILLTHRRIKSCVQLASSERDMESSGVPVITLSSGKVMPVLGMGTFEKVGKGSERERLAILKAIEVGYRYFDTAAAYETEEVLGEAIAEALQLGLIESRDELFISSMLWCTDAHPDRVLLALQNSLRQVFLMQIRLIYICTYQQVHLNIYFQINEFVLCDMYRNLKLEYLNNYSSSPASSTKTAVLSHQROQSCALPISGLLHIFMNKN C. majus plant SEQ. IDGLIHVTLGNMADKYGPIFSFPTGSHRTLVVSSWEMVKECF source; partial- NO. 15TGNNDTAFSNRPIPLAFKTIFYACGGIDSYGLSSVPYGKY length aminoWRELRKVCVHNLLSNQQLLKFRHLIISQVDTSFNKLYELC acid sequenceKNSEDNQGNYPTTTTAAGMVRIDDWLAELSFNVIGRIVCG >chm.CMASTFQSGPKTGAPSRVEQFKEAINEASYFMSTSPVSDNVPMLG 2PF_14984WIDQLTGLTRNMKHCGKKLDLVVESIINDHRQKRRFSRTKGGDEKDDEQDDFIDICLSIMEQPQLPGNNNPSQIPIKSIVLDMIGGGTDTTKLTTIWTLSLLLNNPHVLDKAKQEVDAHFRTKRRSTNDAAAAVVDFDDIRNLVYIQAIIKESMRLYPASPVVERLSGEDCVVGGFHVPAGTRLWANVWKMQRDPKVWDDPLVFRPDRFLSDEQKMVDVRGQNYELLPFGAGRRVCPGVSFSLDLMQLVLTRLILEFEMKSPSGKVDMTATPGLMSYKVIPLDILLTHRRIKPCVQSAASERDMESSGVPVITLGSGKVMPVLGMGTFEKVGKGSERERLAFLKAIEVGYRYFDTAAAYETEEFLGEAIAEALQLGLIKSRDELFITSKLWPCDAHPDLVVPALQNSLRNLKLEYVDLYMLPFPASLKPGKJTMDIPEEDICRMDYRSVWAAMEECQNLGFTKSIGVSNFSCKKLQELMATANIPPAVNQVEMSPAFQQKKLREYCNANNILVSAISVLGSNGTPWGSNAVLGSEVLKKIAMAKGKSVAQVSMRWVYEQGASLVVKSFSEERLRENLNIFDWELTKEDHEKIGEIPQCRI LSAYFLVSPNGPFKSQEELWDDEA*

BisBIA Generating Modifications

Some methods, processes, and systems provided herein describe theproduction of bisbenzylisoquinoline alkaloids (bisBIAs). BisBIAs aredimeric molecules that may be formed by coupling reactions between twoBIA monomers. In examples, bisBIAs may be formed by carbon-oxygencoupling reactions. In other examples, bisBIAs may be formed bycarbon-carbon coupling reactions. In some examples, the bisBIA dimericmolecule is a homodimer, comprising two identical BIA monomers. Inexamples, an engineered host cell may produce one BIA monomer. In theseexamples, the BIA monomers may form homodimers when contacted with oneor more coupling enzymes. In other examples, the bisBIA dimeric moleculeis a heterodimer, comprising two different BIA monomers. For example, abisBIA may be a heterodimer that comprises BIA monomers that areenantiomers of each other. In some examples, an engineered host cell mayproduce two or more BIA monomers. In these examples, the BIA monomersmay form homodimers and heterodimers when contacted with one or morecoupling enzymes.

Some of these methods, processes, and systems that describe theproduction of bisBIAs may comprise an engineered host cell. In someexamples, the engineered host cell may be engineered to produce BIAmonomers which, in turn, may be used as building block molecules forforming bisBIAs. Examples of BIA monomers that may be used to formbisBIAs include coclaurine, N-methylcoclaurine, laudanine,norcoclaurine, norlaudanosoline, 6-O-methyl-norlaudanosoline,3′-hydroxy-N-methylcoclaurine, 3′-hydroxycoclaurine, reticuline,norreticuline, norlaudanine, laudanosine, and papaverine. In particular,engineered host cells may synthesize BIA monomers from norcoclaurine ornorlaudanosoline by expression of heterologous enzymes includingO-methyltransferases, N-methyltransferases, and 3′-hydroxylases.Examples of O-methyltransferases may include norcoclaurine6-O-methyltransferase (6OMT) from Thalicrum flavum, Nelumbo nucifera,Populus euphratica, or another species. Further examples ofO-methyltransferases may include catechol O-methyltransferase (COMT)from Homo sapiens, Mus musculus, Rattus norvegicus, Gorilla gorilla, oranother species. Further examples of N-methyltransferases may includecoclaurine N-methyltransferase (CNMT) from T. flavum, N. nucifera,Aristolochia fimbriata, or another species. Examples of 3′hydroxylasesmay include N-methylcoclaurine 3′-hydroxylase (CYP80B1) fromEschscholzia californica, T. flavum, N. nucifera, or another species.

The engineered host cells may produce either (S) or (R) enantiomers ofany given BIA monomer. Additionally or alternatively, the engineeredhost cells may produce a mixture of both enantiomers. The ratio of (S)and (R) enantiomers may be determined by the substrate and productspecificities of the one or more enzymes that synthesize the BIAmonomers. Alternatively, the amount of each enantiomer present may bemodified by the expression of an additional enzyme or enzymes thatperform the epimerization of one stereoisomer into another, as discussedabove.

These BIA monomers may be fused into a dimeric bisBIA scaffold. Inparticular, the BIA monomers may be fused into a dimeric bisBIA scaffoldutilizing one or more enzymes that are produced by the engineered hostcell. Additionally or alternatively, the BIA monomers may be fused intoa dimeric bisBIA scaffold utilizing one or more enzymes that areprovided to the BIA monomers from a source that is external to theengineered host cell. The one or more enzymes may be used to formcarbon-oxygen and/or carbon-carbon coupling reactions to fuse two BIAmonomers at one, two, or three positions. In some examples, two BIAmonomers may be linked by an ether bridge. In some examples, a directcarbon-carbon bond may be used to connect the two BIA monomers. In someexamples, a bisBIA that is formed by fusing two BIA monomers maycomprise one diphenyl ether linkage. In some examples, two BIA monomersmay be fused to form a bisBIA that comprises two diphenyl etherlinkages. In some examples, a bisBIA that is formed from two BIAmonomers may comprise three diphenyl ether linkages. In some examples,the bisBIA may comprise one diphenyl ether linkage and one benzyl phenylether linkage. In some cases, the bisBIA may comprise one benzyl phenylether linkage and two diphenyl ether linkages.

In examples, the BIA monomers may be contacted with a sufficient amountof the one or more enzymes that may be used to form coupling reactionsto fuse two BIA monomers such that at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 82%, at least 84%, at least86%, at least 88%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, at least 99.7%, or 100% of said BIAmonomers are converted to bisBIAs. The one or more enzymes that may beused to dimerize the BIA monomers into bisBIAs may contact the BIAmonomers in vitro. Additionally, or alternatively, the one or moreenzymes that may be used to dimerize the BIA monomers into bisBIAs maycontact the BIA monomers in vivo. Additionally, the one or more bisBIAdimerizing enzyme may be expressed in a host cell that produces BIAmonomers. Alternatively, the BIA monomers may be provided to theengineered host cell that expresses the bisBIA dimerizing enzyme.Alternatively, the one or more bisBIA dimerizing enzymes may be providedto a cell having BIA monomers within.

In some examples, the bisbenzylisoquinoline alkaloid is a compound ofany one of Formulas Va-Vu:

or a salt thereof, wherein:R^(1a), R^(1b), R^(2a), and R^(2b) are independently selected fromhydrogen and C₁-C₄ alkyl;R^(3a), R^(3b), R^(6a), R^(6b), R^(8a), and R^(8b) are independentlyselected from hydrogen, hydroxy, fluoro, chloro, bromo, carboxaldehyde,acyl, C₁-C₄ alkyl, and C₁-C₄ alkoxy;R^(4a) and R^(5a) are independently selected from hydrogen and C₁-C₄alkyl, or R^(4a) and R^(5a) together form a methylene bridge;R^(4b) and R^(5b) are independently selected from hydrogen and C₁-C₄alkyl, or R^(4b) and R^(5b) together form a methylene bridge; andR^(7a), R^(7b), and R^(9a) are independently selected from hydrogen andC₁-C₄ alkyl.

In some examples, R^(1a) and R^(1b) are each hydrogen; R^(2a) and R^(2b)are each methyl; R^(3a) and R^(3b) are each hydrogen; R^(4a) and R^(5a)are independently hydrogen or methyl; R^(4b) and R^(5b) areindependently hydrogen or methyl, or R^(4b) and R^(5b) together form amethylene bridge; R^(6a), R^(6b), R^(8a), and R^(8b) are each hydrogen;and R^(7a), R^(7b), and R^(9a) are independently hydrogen or methyl.

As illustrated above, the bisBIA compounds of Formulas Va, Vb, and Vdare formed by fusing two BIA monomers using a carbon-oxygen couplingreaction. Additionally, the bisBIA compounds of Formulas Vc, Vf, and Vhare formed by fusing two BIA monomers using both a carbon-oxygencoupling reaction and a carbon-carbon coupling reaction. Further, thebisBIA compounds of Formulas Ve, Vg, Vi, Vj, Vk, Vl, Vm, Vo, Vp, and Vqare formed by fusing two BIA monomers using two carbon-oxygen couplingreactions. The bisBIA compound of Formula Vn is formed by fusing two BIAmonomers using two carbon-oxygen coupling reactions and a carbon-carboncoupling reaction. Additionally, the bisBIA compound of Formula Vr isformed by fusing two BIA monomers using three carbon-oxygen couplingreactions.

The one or more enzymes that may be used to form the coupling reactionsmay include known cytochrome P450s such as Berberis stolonifera CYP80A1or similar cytochrome P450 enzymes from other plants that naturallysynthesize these compounds. Alternatively, the coupling reaction may beperformed by an enzyme that is not a cytochrome P450. The one or moreenzymes that may be used to form the coupling reactions may beengineered to accept non-native substrates. Accordingly, the one or moreenzymes that may be used to form the coupling reactions may be used togenerate non-natural bisBIA molecules. In examples, the one or moreenzymes may fuse a natural BIA monomer with a non-natural BIA monomer toproduce a non-natural bisBIA molecule. In other examples, the one ormore enzymes may fuse two non-natural BIA monomers to produce anon-natural bisBIA molecule. Enzyme engineered strategies may be used toidentify one or more enzymes that may be used to form the couplingreactions that fuse BIA monomers to produce bisBIAs. In examples, enzymeengineering strategies may include site directed mutagenesis, randommutagenesis and screening, DNA shuffling, and screening.

Once bisBIAs are formed, the bisBIAs may be further derivatized ormodified. The bisBIAs may be derivatized or modified utilizing one ormore enzymes that are produced by the engineered host cell. Inparticular, the bisBIAs may be derivatized or modified by contacting thebisBIAs with one or more enzymes that are produced by the engineeredhost cell. Additionally or alternatively, the bisBIAs may be derivatizedor modified by contacting the bisBIAs with one or more enzymes that areprovided to the bisBIAs from a source that is external to the engineeredhost cell. The one or more enzymes that may be used to derivatize ormodify the bisBIAs may be used to perform tailoring reactions. Examplesof tailoring reactions include oxidation, reduction, O-methylation,N-methylation, O-demethylation, acetylation, methylenedioxybridgeformation, and O,O-demethylenation. A bisBIA may be derivatized ormodified using one or more tailoring reactions.

Examples of tailoring reactions are provided in Table 8. In someexamples, tailoring enzymes may be used to catalyze carbon-carboncoupling reactions performed on a bisBIA, or a derivative thereof.Examples of tailoring enzymes that may be used to catalyze carbon-carboncoupling reactions include a Berberine bridge enzyme (BBE) from Papaversomniferum, Eschscholzia californica, Coptis japonica, Berberisstolonifer, Thalictrum flavum, or another species; Salutaridine synthase(SalSyn) from Papaver somniferum or another species; and Corytuberinesynthase (CorSyn) from Coptis japonica or another species. Non-limitingexamples of reactions that can be catalyzed by tailoring enzymes areshown in Scheme 2, wherein R^(a), R^(b), R^(c), and R^(d) areindependently selected from hydrogen, hydroxy, fluoro, chloro, bromo,carboxaldehyde, C₁-C₄ acyl, C₁-C₄ alkyl, and C₁-C₄ alkoxy. In someexamples, R^(a), R^(b), and the carbon atoms to which they are attachedoptionally form a carbocycle or heterocycle. In some examples, R^(c),R^(d), and the carbon atoms to which they are attached optionally form acarbocycle or heterocycle.

In some examples, tailoring enzymes may be used to catalyze oxidationreactions performed on a bisBIA, or a derivative thereof. Examples oftailoring enzymes that may be used to catalyze oxidation reactionsinclude a Tetrahydroprotoberberine oxidase (STOX) from Coptis japonica,Argemone mexicana, Berberis wilsonae, or another species;Dihydrobenzophenanthridine oxidase (DBOX) from Papaver somniferum oranother species; Methylstylopine hydroxylase (MSH) from Papaversomniferum or another species; and Protopine 6-hydroxylase (P6H) fromPapaver somniferum, Eschscholzia californica, or another species.

Tailoring enzymes may also be used to catalyze methylenedioxy bridgeformation reactions performed on a bisBIA, or a derivative thereof.Examples of tailoring enzymes that may be used to catalyzemethylenedioxy bridge formation reactions include a Stylopine synthase(StySyn or STS) from Papaver somniferum, Eschscholzia californica,Argemone mexicana, or another species; Cheilanthifoline synthase (CheSynor CFS) from Papaver somniferum, Eschscholzia californica, Argemonemexicana, or another species; and Canadine synthase (CAS) fromThalictrum flavum, Coptis chinensis, or another species.

In other examples, tailoring enzymes may be used to catalyzeO-methylation reactions performed on a bisBIA, or a derivative thereof.Examples of tailoring enzymes that may be used to catalyze O-methylationreactions include a Norcoclaurine 6-O-methyltransferase (6OMT) fromPapaver somniferum, Thalictrum flavum, Coptis japonica, Papaverbracteatum, or another species; 3′hydroxy-N-methylcoclaurine4′-O-methyltransferase (4′OMT) from Papaver somniferum, Thalictrumflavum, Coptis japonica, Coptis chinensis, or another species;Reticuline 7-O-methyltransferase (7OMT) from Papaver somniferum,Eschscholzia californica, or another species; and Scoulerine9-O-methyltransferase (9OMT) from Papaver somniferum, Thalictrum flavum,Coptis japonica, Coptis chinensis, or another species.

Additionally, tailoring enzymes may be used to catalyze N-methylationreactions performed on a bisBIA, or a derivative thereof. Examples oftailoring enzymes that may be used to catalyze N-methylation reactionsinclude Coclaurine N-methyltransferase (CNMT) from Papaver somniferum,Thalictrum flavum, Coptis japonica, or another species;Tetrahydroprotoberberine N-methyltransferase (TNMT) from Papaversomniferum, Eschscholzia californica, Papaver bracteatum, or anotherspecies.

Further, tailoring enzymes may be used to catalyze O-demethylationreactions performed on a bisBIA, or a derivative thereof. Examples oftailoring enzymes that may be used to catalyze O-demethylation reactionsinclude Thebaine demethylase (T6ODM) from Papaver somniferum or anotherspecies; and Codeine demethylase (CODM) from Papaver somniferum, oranother species.

Tailoring enzymes may also be used to catalyze reduction reactionsperformed on a bisBIA, or a derivative thereof. Examples of tailoringenzymes that may be used to catalyze reduction reactions includeSalutaridine reductase (SalR) from Papaver somniferum, Papaverbracteatum, or another species; Codeinone reductase (COR) from Papaversomniferum or another species; and Sanguinarine reductase (SanR) fromEschscholzia californica or another species. In other examples,tailoring enzymes may be used to catalyze acetylation reactionsperformed on a bisBIA, or a derivative thereof. An example of atailoring enzyme that may be used to catalyze acetylation reactionsincludes Salutaridine acetyltransferase (SalAT) from Papaver somniferumor another species.

O-Demethylation Modifications

Some methods, processes, and systems provided herein describe theconversion of a first benzylisoquinoline alkaloid to a secondbenzylisoquinoline alkaloid by the removal of an O-linked methyl group.Some of these methods, processes, and systems may comprise an engineeredhost cell. In some examples, the conversion of a firstbenzylisoquinoline alkaloid to a second benzylisoquinoline alkaloid is akey step in the conversion of a substrate to a nor-opioids ornal-opioids. In some examples, the conversion of a first alkaloid to asecond alkaloid comprises a demethylase reaction.

FIG. 23 illustrates an enzyme having opioid 3-O-demethylase activity, inaccordance with embodiments of the invention. Specifically, the enzymemay act on any morphinan alkaloid structure to remove the methyl groupfrom the oxygen bound to carbon 3.

Examples of amino acid sequences of ODM enzymes are set forth in Table3. An amino acid sequence for an ODM that is utilized in converting afirst alkaloid to a second alkaloid may be 75% or more identical to agiven amino acid sequence as listed in Table 3. For example, an aminoacid sequence for such an epimerase may comprise an amino acid sequencethat is at least 75% or more, 80% or more, 81% or more, 82% or more, 83%or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% ormore, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more,94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99%or more identical to an amino acid sequence as provided herein.Additionally, in certain embodiments, an “identical” amino acid sequencecontains at least 80%-99% identity at the amino acid level to thespecific amino acid sequence. In some cases an “identical” amino acidsequence contains at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94% and more in certain cases, at least 95%, 96%, 97%, 98% and 99%identity, at the amino acid level. In some cases, the amino acidsequence may be identical but the DNA sequence is altered such as tooptimize codon usage for the host organism, for example.

An engineered host cell may be provided that produces an ODM thatconverts a first alkaloid to a second alkaloid, wherein the ODMcomprises a given amino acid sequence as listed in Table 3. Anengineered host cell may be provided that produces one or more ODMenzymes. The ODM that is produced within the engineered host cell may berecovered and purified so as to form a biocatalyst. The process mayinclude contacting the first alkaloid with an ODM in an amountsufficient to convert said first alkaloid to a second alkaloid. Inexamples, the first alkaloid may be contacted with a sufficient amountof the one or more enzymes such that at least 5% of said first alkaloidis converted to a second alkaloid. In further examples, the firstalkaloid may be contacted with a sufficient amount of the one or moreenzymes such that at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 82%, at least 84%, at least 86%, at least88%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, at least 99.5%, at least 99.7%, or 100% of said first alkaloid isconverted to a second alkaloid.

The one or more enzymes that may be used to convert a first alkaloid toa second alkaloid may contact the first alkaloid in vitro. Additionally,or alternatively, the one or more enzymes that may be used to convert afirst alkaloid to a second alkaloid may contact the first alkaloid invivo. In some examples, the one or more enzymes that may be used toconvert a first alkaloid to a second alkaloid may be provided to a cellhaving the first alkaloid within. In some examples, the one or moreenzymes that may be used to convert a first alkaloid to a secondalkaloid may be produced within an engineered host cell.

In some examples, the methods provide for engineered host cells thatproduce an alkaloid product, wherein the O-demethylation of a substrateto a product may comprise a key step in the production of an alkaloidproduct. In some examples, the alkaloid produced is a nor-opioid or anal-opioid. In still other embodiments, the alkaloid produced is derivedfrom a nor-opioid or a nal-opioid. In another embodiment, a firstalkaloid is an intermediate toward the product of the engineered hostcell. In still other embodiments, the alkaloid product is selected fromthe group consisting of morphine, oxymorphine, oripavine, hydromorphone,dihydromorphine, 14-hydroxymorphine, morphinone, and14-hydroxymorphinone.

In some examples, the substrate alkaloid is an opioid selected from thegroup consisting of codeine, oxycodone, thebaine, hydrocodone,dihydrocodeine, 14-hydroxycodeine, codeinone, and 14-hydroxycodeinone.

N-Demethylation Modifications

Some methods, processes, and systems provided herein describe theconversion of a first alkaloid to a second alkaloid by the removal of anN-linked methyl group. Some of these methods, processes, and systems maycomprise an engineered host cell. In some examples, the conversion of afirst alkaloid to a second alkaloid is a key step in the conversion of asubstrate to a nor-opioids or nal-opioids. In some examples, theconversion of a first alkaloid to a second alkaloid comprises ademethylase reaction.

FIG. 24 illustrates an enzyme having opioid N-demethylase activity, inaccordance with embodiments of the invention. Specifically, the enzymemay act on any morphinan alkaloid structure to remove the methyl groupfrom the nitrogen.

Examples of an amino acid sequence of an N-demethylase enzyme that maybe used to perform the conversion a first alkaloid to a second alkaloidare provided in Table 4. An amino acid sequence for an NDM that isutilized in converting a first alkaloid to a second alkaloid may be 75%or more identical to a given amino acid sequence as listed in Table 4.For example, an amino acid sequence for such an epimerase may comprisean amino acid sequence that is at least 75% or more, 80% or more, 81% ormore, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more,87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% ormore, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more,98% or more, or 99% or more identical to an amino acid sequence asprovided herein. Additionally, in certain embodiments, an “identical”amino acid sequence contains at least 80%-99% identity at the amino acidlevel to the specific amino acid sequence. In some cases an “identical”amino acid sequence contains at least about 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94% and more in certain cases, at least 95%, 96%,97%, 98% and 99% identity, at the amino acid level. In some cases, theamino acid sequence may be identical but the DNA sequence is alteredsuch as to optimize codon usage for the host organism, for example.

An engineered host cell may be provided that produces an NDM thatconverts a first alkaloid to a second alkaloid, wherein the NDMcomprises an amino acid sequence as listed in Table 4. An engineeredhost cell may be provided that produces one or more NDM enzymes. The NDMthat is produced within the engineered host cell may be recovered andpurified so as to form a biocatalyst. The process may include contactingthe first alkaloid with an NDM in an amount sufficient to convert saidfirst alkaloid to a second alkaloid. In examples, the first alkaloid maybe contacted with a sufficient amount of the one or more enzymes suchthat at least 5% of said first alkaloid is converted to a secondalkaloid. In further examples, the first alkaloid may be contacted witha sufficient amount of the one or more enzymes such that at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 82%, atleast 84%, at least 86%, at least 88%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%,or 100% of said first alkaloid is converted to a second alkaloid.

The one or more enzymes that may be used to convert a first alkaloid toa second alkaloid may contact the first alkaloid in vitro. Additionally,or alternatively, the one or more enzymes that may be used to convert afirst alkaloid to a second alkaloid may contact the first alkaloid invivo. In some examples, the one or more enzymes that may be used toconvert a first alkaloid to a second alkaloid may be provided to a cellhaving the first alkaloid within. In some examples, the one or moreenzymes that may be used to convert a first alkaloid to a secondalkaloid may be produced within an engineered host cell.

In some examples, the methods provide for engineered host cells thatproduce an alkaloid product, wherein the N-demethylation of a substrateto a product may comprise a key step in the production of an alkaloidproduct. In some examples, the alkaloid produced is a nor-opioid or anal-opioid. In still other embodiments, the alkaloid produced is derivedfrom a nor-opioid or a nal-opioid. In another embodiment, a firstalkaloid is an intermediate toward the product of the engineered hostcell. In still other embodiments, the alkaloid product is selected fromthe group consisting of norcodeine, noroxycodone, northebaine,norhydrocodone, nordihydro-codeine, nor-14-hydroxy-codeine,norcodeinone, nor-14-hydroxy-codeinone, normorphine, noroxymorphone,nororipavine, norhydro-morphone, nordihydro-morphine,nor-14-hydroxy-morphine, normorphinone, and nor-14-hydroxy-morphinone.

In some examples, the substrate alkaloid is an opioid selected from thegroup consisting of codeine, oxycodone, thebaine, hydrocodone,dihydrocodeine, 14-hydroxycodeine, codeinone, and 14-hydroxycodeinone,morphine, oxymorphone, oripavine, hydromorphone, dihydromorphine,14-hydroxy-morphine, morphinone, or 14-hydroxy-morphinone.

N-Methyltransferase Modifications

Some methods, processes, and systems provided herein describe theconversion of a first alkaloid to a second alkaloid by the addition ofan N-linked sidechain group. Some methods, processes, and systemsprovided herein describe the conversion of a first alkaloid to a secondalkaloid by the transfer of a sidechain group from a cosubstrate to thefirst alkaloid. Some of these methods, processes, and systems maycomprise an engineered host cell. In some examples, the conversion of afirst alkaloid to a second alkaloid is a key step in the conversion of asubstrate to a nal-opioid. In some examples, the conversion of a firstalkaloid to a second alkaloid comprises a methyltransferase reaction.

FIG. 25 illustrates an enzyme having N-methyltransferase activity, inaccordance with embodiments of the invention. Specifically, the enzymemay act on any morphinan alkaloid structure to add a methyl group orother carbon moiety to the nitrogen. S-Adenosyl methionine (SAM) may actas the donor of the functional group (methyl, allyl, cyclopropylmethyl,or other).

Examples of amino acid sequences of NMT enzymes are set forth in Table5. An amino acid sequence for an NMT that is utilized in converting afirst alkaloid to a second alkaloid may be 75% or more identical to agiven amino acid sequence as listed in Table 5. For example, an aminoacid sequence for such an epimerase may comprise an amino acid sequencethat is at least 75% or more, 80% or more, 81% or more, 82% or more, 83%or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% ormore, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more,94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99%or more identical to an amino acid sequence as provided herein.Additionally, in certain embodiments, an “identical” amino acid sequencecontains at least 80%-99% identity at the amino acid level to thespecific amino acid sequence. In some cases an “identical” amino acidsequence contains at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94% and more in certain cases, at least 95%, 96%, 97%, 98% and 99%identity, at the amino acid level. In some cases, the amino acidsequence may be identical but the DNA sequence is altered such as tooptimize codon usage for the host organism, for example.

An engineered host cell may be provided that produces an NMT thatconverts a first alkaloid to a second alkaloid, wherein the NMTcomprises an amino acid sequence as provided in Table 5. An engineeredhost cell may be provided that produces one or more NMT enzymes. The NMTthat is produced within the engineered host cell may be recovered andpurified so as to form a biocatalyst. The process may include contactingthe first alkaloid with an NMT in an amount sufficient to convert saidfirst alkaloid to a second alkaloid. In examples, the first alkaloid maybe contacted with a sufficient amount of the one or more enzymes suchthat at least 5% of said first alkaloid is converted to a secondalkaloid. In further examples, the first alkaloid may be contacted witha sufficient amount of the one or more enzymes such that at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 82%, atleast 84%, at least 86%, at least 88%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%,or 100% of said first alkaloid is converted to a second alkaloid.

The one or more enzymes that may be used to convert a first alkaloid toa second alkaloid may contact the first alkaloid in vitro. Additionally,or alternatively, the one or more enzymes that may be used to convert afirst alkaloid to a second alkaloid may contact the first alkaloid invivo. In some examples, the one or more enzymes that may be used toconvert a first alkaloid to a second alkaloid may be provided to a cellhaving the first alkaloid within. In some examples, the one or moreenzymes that may be used to convert a first alkaloid to a secondalkaloid may be produced within an engineered host cell.

In some examples, the methods provide for engineered host cells thatproduce an alkaloid product, wherein the N-methyltransferase of asubstrate to a product may comprise a key step in the production of analkaloid product. In some examples, the alkaloid produced is anal-opioid. In still other embodiments, the alkaloid produced is derivedfrom a nor-opioid or a nal-opioid. In another embodiment, a firstalkaloid is an intermediate toward the product of the engineered hostcell. In still other embodiments, the alkaloid product is selected fromthe group including naloxone, naltrexone, and nalmefene.

In some examples, the substrate alkaloid is an opioid selected from thegroup consisting of norcodeine, noroxycodone, northebaine,norhydrocodone, nordihydro-codeine, nor-14-hydroxy-codeine,norcodeinone, nor-14-hydroxy-codeinone, normorphine, noroxymorphone,nororipavine, norhydro-morphone, nordihydro-morphine,nor-14-hydroxy-morphine, normorphinone, and nor-14-hydroxy-morphinone.In some examples, the cosubstrate is S-adenosylmethionine,Allyl-S-adenosylmethionine, or cyclopropylmethyl-S-adenosylmethionine.

Heterologous Coding Sequences

In some instances, the engineered host cells harbor one or moreheterologous coding sequences (such as two or more, three or more, fouror more, five or more) which encode activity(ies) that enable theengineered host cells to produce desired enzymes of interest and/or BIAsof interest, e.g., as described herein. As used herein, the term“heterologous coding sequence” is used to indicate any polynucleotidethat codes for, or ultimately codes for, a peptide or protein or itsequivalent amino acid sequence, e.g., an enzyme, that is not normallypresent in the host organism and may be expressed in the host cell underproper conditions. As such, “heterologous coding sequences” includesmultiple copies of coding sequences that are normally present in thehost cell, such that the cell is expressing additional copies of acoding sequence that are not normally present in the cells. Theheterologous coding sequences may be RNA or any type thereof, e.g.,mRNA, DNA or any type thereof, e.g., cDNA, or a hybrid of RNA/DNA.Coding sequences of interest include, but are not limited to,full-length transcription units that include such features as the codingsequence, introns, promoter regions, 3′-UTRs, and enhancer regions.

The engineered host cells may also be modified to possess one or moregenetic alterations to accommodate the heterologous coding sequences.Alterations of the native host genome include, but are not limited to,modifying the genome to reduce or ablate expression of a specificprotein that may interfere with the desired pathway. The presence ofsuch native proteins may rapidly convert one of the intermediates orfinal products of the pathway into a metabolite or other compound thatis not usable in the desired pathway. Thus, if the activity of thenative enzyme were reduced or altogether absent, the producedintermediates would be more readily available for incorporation into thedesired product.

Heterologous coding sequences include but are not limited to sequencesthat encode enzymes, either wild-type or equivalent sequences, that arenormally responsible for the production of BIAs of interest in plants.In some cases, the enzymes for which the heterologous sequences code maybe any of the enzymes in the 1-BIA pathway, and may be from anyconvenient source. The choice and number of enzymes encoded by theheterologous coding sequences for the particular synthetic pathway maybe selected based upon the desired product. In certain embodiments, thehost cells may include 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, 13 or more, 14 or more, or even 15 or moreheterologous coding sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 heterologous coding sequences.

As used herein, the term “heterologous coding sequences” also includesthe coding portion of the peptide or enzyme, i.e., the cDNA or mRNAsequence, of the peptide or enzyme, as well as the coding portion of thefull-length transcriptional unit, i.e., the gene including introns andexons, as well as “codon optimized” sequences, truncated sequences orother forms of altered sequences that code for the enzyme or code forits equivalent amino acid sequence, provided that the equivalent aminoacid sequence produces a functional protein. Such equivalent amino acidsequences may have a deletion of one or more amino acids, with thedeletion being N-terminal, C-terminal, or internal. Truncated forms areenvisioned as long as they have the catalytic capability indicatedherein. Fusions of two or more enzymes are also envisioned to facilitatethe transfer of metabolites in the pathway, provided that catalyticactivities are maintained.

Operable fragments, mutants, or truncated forms may be identified bymodeling and/or screening. In some cases, this is achieved by deletionof, for example, N-terminal, C-terminal, or internal regions of theprotein in a step-wise fashion, followed by analysis of the resultingderivative with regard to its activity for the desired reaction comparedto the original sequence. If the derivative in question operates in thiscapacity, it is considered to constitute an equivalent derivative of theenzyme proper.

In examples, some heterologous proteins may show occurrences where theyare incorrectly processed when expressed in a recombinant host. Forexample, plant proteins such as cytochrome P450 enzymes expressed inmicrobial production hosts may have occurrences of incorrect processing.In particular, salutaridine synthase may undergo N-linked glycosylationwhen heterologously expressed in yeast. This N-linked glycosylation maynot be observed in plants, which may be indicative of incorrectN-terminal sorting of the nascent SalSyn transcript so as to reduce theactivity of the enzyme in the heterologous microbial host. In suchexamples, protein engineering directed at correcting N-terminal sortingof the nascent transcript so as to remove the N-linked glycosylationpattern may result in improved activity of the salutaridine synthaseenzyme in the recombinant production host. This is explained further inExample 8 below.

Some aspects of the invention also relate to heterologous codingsequences that code for amino acid sequences that are equivalent to thenative amino acid sequences for the various enzymes. An amino acidsequence that is “equivalent” is defined as an amino acid sequence thatis not identical to the specific amino acid sequence, but rathercontains at least some amino acid changes (deletions, substitutions,inversions, insertions, etc.) that do not essentially affect thebiological activity of the protein as compared to a similar activity ofthe specific amino acid sequence, when used for a desired purpose. Thebiological activity refers to, in the example of an epimerase, itscatalytic activity. Equivalent sequences are also meant to include thosewhich have been engineered and/or evolved to have properties differentfrom the original amino acid sequence. Mutable properties of interestinclude catalytic activity, substrate specificity, selectivity,stability, solubility, localization, etc.

In some instances, the expression of each type of enzyme is increasedthrough additional gene copies (i.e., multiple copies), which increasesintermediate accumulation and/or BIA of interest production. Someembodiments of the invention include increased BIA of interestproduction in a host cell through simultaneous expression of multiplespecies variants of a single or multiple enzymes. In some cases,additional gene copies of a single or multiple enzymes are included inthe host cell. Any convenient methods may be utilized including multiplecopies of a heterologous coding sequence for an enzyme in the host cell.

In some examples, the engineered host cell includes multiple copies of aheterologous coding sequence for an enzyme, such as 2 or more, 3 ormore, 4 or more, 5 or more, or even 10 or more copies. In certainembodiments, the engineered host cell includes multiple copies ofheterologous coding sequences for one or more enzymes, such as multiplecopies of two or more, three or more, four or more, etc. In some cases,the multiple copies of the heterologous coding sequence for an enzymeare derived from two or more different source organisms as compared tothe host cell. For example, the engineered host cell may includemultiple copies of one heterologous coding sequence, where each of thecopies is derived from a different source organism. As such, each copymay include some variations in explicit sequences based on inter-speciesdifferences of the enzyme of interest that is encoded by theheterologous coding sequence.

The engineered host cell medium may be sampled and monitored for theproduction of BIAs of interest. The BIAs of interest may be observed andmeasured using any convenient methods. Methods of interest include, butare not limited to, LC-MS methods (e.g., as described herein) where asample of interest is analyzed by comparison with a known amount of astandard compound. Additionally, there are other ways that BIAs ofinterest may be observed and/or measured. Examples of alternative waysof observing and/or measuring BIAs include GC-MS, UV-vis spectroscopy,NMR, LC-NMR, LC-UV, TLC, capillary electrophoresis, among others.Identity may be confirmed, e.g., by m/z and MS/MS fragmentationpatterns, and quantitation or measurement of the compound may beachieved via LC trace peaks of know retention time and/or EIC MS peakanalysis by reference to corresponding LC-MS analysis of a known amountof a standard of the compound.

Additionally, a culture of the engineered host cell may be sampled andmonitored for the production of enzymes of interest, such as a CYP-CORenzyme. The enzymes of interest may be observed and measured using anyconvenient methods. Methods of interest include enzyme activity assays,polyacrylamide gel electrophoresis, carbon monoxide spectroscopy, andwestern blot analysis.

Methods Methods for Culturing Host Cells for BIA Production

As summarized above, some aspects of the invention include methods ofpreparing nor-opioid and nal-opioid BIAs of interest. Additionally, someaspects of the invention include methods of preparing enzymes ofinterest. As such, some aspects of the invention include culturing anengineered host cell under conditions in which the one or more host cellmodifications (e.g., as described herein) are functionally expressedsuch that the cell converts starting compounds of interest into productnor-opioid and/or nal-opioid BIAs of interest. Also provided are methodsthat include culturing an engineered host cell under conditions suitablefor protein production such that one or more heterologous codingsequences are functionally expressed and convert starting compounds ofinterest into product enzymes or nor-opioid and/or nal-opioid BIAs ofinterest. In examples, the method is a method of preparing a nor-opioidand/or nal-opioid BIA of interest that includes culturing an engineeredhost cell (e.g., as described herein); adding a starting compound to thecell culture; and recovering the nor-opioid and/or nal-opioid from thecell culture. In some examples, the method is a method of preparing anenzyme that includes culturing an engineered host cell (e.g., asdescribed herein); adding a starting compound to the cell culture; andrecovering the enzyme from the cell culture.

Fermentation media may contain suitable carbon substrates. The source ofcarbon suitable to perform the methods of this disclosure may encompassa wide variety of carbon containing substrates. Suitable substrates mayinclude, without limitation, monosaccharides (e.g., glucose, fructose,galactose, xylose), oligosaccharides (e.g., lactose, sucrose,raffinose), polysaccharides (e.g., starch, cellulose), or a combinationthereof. In some cases, unpurified mixtures from renewable feedstocksmay be used (e.g., cornsteep liquor, sugar beet molasses, barley malt).In some cases, the carbon substrate may be a one-carbon substrate (e.g.,methanol, carbon dioxide) or a two-carbon substrate (e.g., ethanol). Inother cases, other carbon containing compounds may be utilized, forexample, methylamine, glucosamine, and amino acids.

Any convenient methods of culturing engineered host cells may beemployed for producing the nor-opioid and/or nal-opioid BIAs ofinterest. The particular protocol that is employed may vary, e.g.,depending on the engineered host cell, the heterologous codingsequences, the enzymes of interest, the nor-opioid and/or nal-opioidBIAs of interest, etc. The cells may be present in any convenientenvironment, such as an environment in which the cells are capable ofexpressing one or more functional heterologous enzymes. In someembodiments, the cells are cultured under conditions that are conduciveto enzyme expression and with appropriate substrates available to allowproduction of nor-opioid and/or nal-opioid BIAs of interest in vivo. Insome embodiments, the functional enzymes are extracted from theengineered host for production of nor-opioid and/or nal-opioid BIAs ofinterest under in vitro conditions. In some instances, the engineeredhost cells are placed back into a multicellular host organism. Theengineered host cells are in any phase of growth, including, but notlimited to, stationary phase and log-growth phase, etc. In addition, thecultures themselves may be continuous cultures or they may be batchcultures.

Cells may be grown in an appropriate fermentation medium at atemperature between 14-40° C. Cells may be grown with shaking at anyconvenient speed (e.g., 200 rpm). Cells may be grown at a suitable pH.Suitable pH ranges for the fermentation may be between pH 5-9.Fermentations may be performed under aerobic, anaerobic, or microaerobicconditions. Any suitable growth medium may be used. Suitable growthmedia may include, without limitation, common commercially preparedmedia such as synthetic defined (SD) minimal media or yeast extractpeptone dextrose (YEPD) rich media. Any other rich, defined, orsynthetic growth media appropriate to the microorganism may be used.

Cells may be cultured in a vessel of essentially any size and shape.Examples of vessels suitable to perform the methods of this disclosuremay include, without limitation, multi-well shake plates, test tubes,flasks (baffled and non-baffled), and bioreactors. The volume of theculture may range from 10 microliters to greater than 10,000 liters.

The addition of agents to the growth media that are known to modulatemetabolism in a manner desirable for the production of alkaloids may beincluded. In a non-limiting example, cyclic adenosine 2′3′-monophosphatemay be added to the growth media to modulate catabolite repression.

Any convenient cell culture conditions for a particular cell type may beutilized. In certain embodiments, the host cells that include one ormore modifications are cultured under standard or readily optimizedconditions, with standard cell culture media and supplements. As oneexample, standard growth media when selective pressure for plasmidmaintenance is not required may contain 20 g/L yeast extract, 10 g/Lpeptone, and 20 g/L dextrose (YPD). Host cells containing plasmids aregrown in synthetic complete (SC) media containing 1.7 g/L yeast nitrogenbase, 5 g/L ammonium sulfate, and 20 g/L dextrose supplemented with theappropriate amino acids required for growth and selection. Alternativecarbon sources which may be useful for inducible enzyme expressioninclude, but are not limited to, sucrose, raffinose, and galactose.Cells are grown at any convenient temperature (e.g., 30° C.) withshaking at any convenient rate (e.g., 200 rpm) in a vessel, e.g., intest tubes or flasks in volumes ranging from 1-1000 mL, or larger, inthe laboratory.

Culture volumes may be scaled up for growth in larger fermentationvessels, for example, as part of an industrial process. The industrialfermentation process may be carried out under closed-batch, fed-batch,or continuous chemostat conditions, or any suitable mode offermentation. In some cases, the cells may be immobilized on a substrateas whole cell catalysts and subjected to fermentation conditions foralkaloid production.

Batch fermentation is a closed system, in which the composition of themedium is set at the beginning of the fermentation and not alteredduring the fermentation process. The desired organism(s) are inoculatedinto the medium at the beginning of the fermentation. In some instances,the batch fermentation is run with alterations made to the system tocontrol factors such as pH and oxygen concentration (but not carbon). Inthis type of fermentation system, the biomass and metabolitecompositions of the system change continuously over the course of thefermentation. Cells typically proceed through a lag phase, then to a logphase (high growth rate), then to a stationary phase (growth ratereduced or halted), and eventually to a death phase (if left untreated).

A continuous fermentation is an open system, in which a definedfermentation medium is added continuously to the bioreactor and an equalamount of fermentation media is continuously removed from the vessel forprocessing. Continuous fermentation systems are generally operated tomaintain steady state growth conditions, such that cell loss due tomedium being removed must be balanced by the growth rate in thefermentation. Continuous fermentations are generally operated atconditions where cells are at a constant high cell density. Continuousfermentations allow for the modulation of one or more factors thataffect target product concentration and/or cell growth.

The liquid medium may include, but is not limited to, a rich orsynthetic defined medium having an additive component described above.Media components may be dissolved in water and sterilized by heat,pressure, filtration, radiation, chemicals, or any combination thereof.Several media components may be prepared separately and sterilized, andthen combined in the fermentation vessel. The culture medium may bebuffered to aid in maintaining a constant pH throughout thefermentation.

Process parameters including temperature, dissolved oxygen, pH,stirring, aeration rate, and cell density may be monitored or controlledover the course of the fermentation. For example, temperature of afermentation process may be monitored by a temperature probe immersed inthe culture medium. The culture temperature may be controlled at the setpoint by regulating the jacket temperature. Water may be cooled in anexternal chiller and then flowed into the bioreactor control tower andcirculated to the jacket at the temperature required to maintain the setpoint temperature in the vessel.

Additionally, a gas flow parameter may be monitored in a fermentationprocess. For example, gases may be flowed into the medium through asparger. Gases suitable for the methods of this disclosure may includecompressed air, oxygen, and nitrogen. Gas flow may be at a fixed rate orregulated to maintain a dissolved oxygen set point.

The pH of a culture medium may also be monitored. In examples, the pHmay be monitored by a pH probe that is immersed in the culture mediuminside the vessel. If pH control is in effect, the pH may be adjusted byacid and base pumps which add each solution to the medium at therequired rate. The acid solutions used to control pH may be sulfuricacid or hydrochloric acid. The base solutions used to control pH may besodium hydroxide, potassium hydroxide, or ammonium hydroxide.

Further, dissolved oxygen may be monitored in a culture medium by adissolved oxygen probe immersed in the culture medium. If dissolvedoxygen regulation is in effect, the oxygen level may be adjusted byincreasing or decreasing the stirring speed. The dissolved oxygen levelmay also be adjusted by increasing or decreasing the gas flow rate. Thegas may be compressed air, oxygen, or nitrogen.

Stir speed may also be monitored in a fermentation process. In examples,the stirrer motor may drive an agitator. The stirrer speed may be set ata consistent rpm throughout the fermentation or may be regulateddynamically to maintain a set dissolved oxygen level.

Additionally, turbidity may be monitored in a fermentation process. Inexamples, cell density may be measured using a turbidity probe.Alternatively, cell density may be measured by taking samples from thebioreactor and analyzing them in a spectrophotometer. Further, samplesmay be removed from the bioreactor at time intervals through a sterilesampling apparatus. The samples may be analyzed for alkaloids producedby the host cells. The samples may also be analyzed for othermetabolites and sugars, the depletion of culture medium components, orthe density of cells.

In another example, a feed stock parameter may be monitored during afermentation process. In particular, feed stocks including sugars andother carbon sources, nutrients, and cofactors that may be added intothe fermentation using an external pump. Other components may also beadded during the fermentation including, without limitation, anti-foam,salts, chelating agents, surfactants, and organic liquids.

Any convenient codon optimization techniques for optimizing theexpression of heterologous polynucleotides in host cells may be adaptedfor use in the subject host cells and methods, see e.g., Gustafsson C.,et al. (2004) Trends Biotechnol, 22, 346-353, which is incorporated byreference in its entirety.

The subject method may also include adding a starting compound to thecell culture. Any convenient methods of addition may be adapted for usein the subject methods. The cell culture may be supplemented with asufficient amount of the starting materials of interest (e.g., asdescribed herein), e.g., an amount in the mM to μM range such as betweenabout 1-5 mM of a starting compound. It is understood that the amount ofstarting material added, the timing and rate of addition, the form ofmaterial added, etc., may vary according to a variety of factors. Thestarting material may be added neat or pre-dissolved in a suitablesolvent (e.g., cell culture media, water, or an organic solvent). Thestarting material may be added in concentrated form (e.g., 10× overdesired concentration) to minimize dilution of the cell culture mediumupon addition. The starting material may be added in one or morebatches, or by continuous addition over an extended period of time(e.g., hours or days).

Methods for Isolating Products from the Fermentation Medium

The subject methods may also include recovering the nor-opioid and/ornal-opioid BIAs of interest from the cell culture. Any convenientmethods of separation and isolation (e.g., chromatography methods orprecipitation methods) may be adapted for use in the subject methods torecover the nor-opioid and/or nal-opioid BIAs of interest from the cellculture. Filtration methods may be used to separate soluble frominsoluble fractions of the cell culture. In some cases, liquidchromatography methods (e.g., reverse phase HPLC, size exclusion, ornormal phase chromatography) may be used to separate the BIA of interestfrom other soluble components of the cell culture. In some cases,extraction methods (e.g., liquid extraction, pH based purification,solid phase extraction, affinity chromatography, ion exchange, etc.) maybe used to separate the nor-opioid and/or nal-opioid BIAs of interestfrom other components of the cell culture.

The produced alkaloids may be isolated from the fermentation mediumusing methods known in the art. A number of recovery steps may beperformed immediately after (or in some instances, during) thefermentation for initial recovery of the desired product. Through thesesteps, the alkaloids (e.g., nor-opioids or nal-opioids) may be separatedfrom the cells, cellular debris and waste, and other nutrients, sugars,and organic molecules may remain in the spent culture medium. Thisprocess may be used to yield a nor-opioid or nal-opioid-enrichedproduct.

In an example, a product stream having a nor-opioid or nal-opioidproduct is formed by providing engineered yeast cells and a feedstockincluding nutrients and water to a batch reactor. In particular, theengineered yeast cells may be subjected to fermentation by incubatingthe engineered yeast cells for a time period of at least about 5 minutesto produce a solution comprising the nor-opioid or nal-opioid productand cellular material. Once the engineered yeast cells have beensubjected to fermentation, at least one separation unit may be used toseparate the nor-opioid or nal-opioid product from the cellular materialto provide the product stream comprising the nor-opioid or nal-opioidproduct. In particular, the product stream may include the nor-opioid ornal-opioid product as well as additional components, such as a clarifiedyeast culture medium. Additionally, a nor-opioid or nal-opioid productmay comprise one or more nor-opioids or nal-opioids of interest, such asone or more nor-opioid or nal-opioid compounds.

Different methods may be used to remove cells from a bioreactor mediumthat include an enzyme and/or nor-opioid or nal-opioid of interest. Inexamples, cells may be removed by sedimentation over time. This processof sedimentation may be accelerated by chilling or by the addition offining agents such as silica. The spent culture medium may then besiphoned from the top of the reactor or the cells may be decanted fromthe base of the reactor. Alternatively, cells may be removed byfiltration through a filter, a membrane, or other porous material. Cellsmay also be removed by centrifugation, for example, by continuous flowcentrifugation or by using a continuous extractor.

Different methods may be used to remove cells from a bioreactor mediumthat include a BIA of interest such as naloxone or naltrexone. Inexamples, cells may be removed by sedimentation over time. This processof sedimentation may be accelerated by chilling or by the addition offining agents such as silica. The spent culture medium may then besiphoned from the top of the reactor or the cells may be decanted fromthe base of the reactor. Alternatively, cells may be removed byfiltration through a filter, a membrane, or other porous material. Cellsmay also be removed by centrifugation, for example, by continuous flowcentrifugation or by using a continuous extractor.

If some valuable nor-opioid and/or nal-opioid BIAs of interest arepresent inside the cells, the cells may be permeabilized or lysed andthe cell debris may be removed by any of the methods described above.Agents used to permeabilize the cells may include, without limitation,organic solvents (e.g., DMSO) or salts (e.g., lithium acetate). Methodsto lyse the cells may include the addition of surfactants such as sodiumdodecyl sulfate, or mechanical disruption by bead milling or sonication.

Nor-opioid and/or nal-opioid BIAs of interest may be extracted from theclarified spent culture medium through liquid-liquid extraction by theaddition of an organic liquid that is immiscible with the aqueousculture medium. In examples, the use of liquid-liquid extraction may beused in addition to other processing steps. Examples of suitable organicliquids include, but are not limited to, isopropyl myristate, ethylacetate, chloroform, butyl acetate, methylisobutyl ketone, methyloleate, toluene, oleyl alcohol, ethyl butyrate. The organic liquid maybe added to as little as 10% or as much as 100% of the volume of aqueousmedium.

In some cases, the organic liquid may be added at the start of thefermentation or at any time during the fermentation. This process ofextractive fermentation may increase the yield of nor-opioid and/ornal-opioid BIAs of interest from the host cells by continuously removingnor-opioids and/or nal-opioids to the organic phase.

Agitation may cause the organic phase to form an emulsion with theaqueous culture medium. Methods to encourage the separation of the twophases into distinct layers may include, without limitation, theaddition of a demulsifier or a nucleating agent, or an adjustment of thepH. The emulsion may also be centrifuged to separate the two phases, forexample, by continuous conical plate centrifugation.

Alternatively, the organic phase may be isolated from the aqueousculture medium so that it may be physically removed after extraction.For example, the solvent may be encapsulated in a membrane.

In examples, nor-opioid and/or nal-opioid BIAs of interest may beextracted from a fermentation medium using adsorption methods. Inexamples, nor-opioids or nal-opioids of interest may be extracted fromclarified spent culture medium by the addition of a resin such asAmberlite® XAD4 or another agent that removes nor-opioids or nal-opioidsby adsorption. The nor-opioids or nal-opioids of interest may then bereleased from the resin using an organic solvent. Examples of suitableorganic solvents include, but are not limited to, methanol, ethanol,ethyl acetate, or acetone.

Nor-opioids or nal-opioids of interest may also be extracted from afermentation medium using filtration. At high pH, the nor-opioids ornal-opioids of interest may form a crystalline-like precipitate in thebioreactor. This precipitate may be removed directly by filtrationthrough a filter, membrane, or other porous material. The precipitatemay also be collected by centrifugation and/or decantation.

The extraction methods described above may be carried out either in situ(in the bioreactor) or ex situ (e.g., in an external loop through whichmedia flows out of the bioreactor and contacts the extraction agent,then is recirculated back into the vessel). Alternatively, theextraction methods may be performed after the fermentation is terminatedusing the clarified medium removed from the bioreactor vessel.

Methods for Purifying Products from Alkaloid-Enriched Solutions

Subsequent purification steps may involve treating the post-fermentationsolution enriched with nor-opioid or nal-opioid product(s) of interestusing methods known in the art to recover individual product species ofinterest to high purity.

In one example, nor-opioids or nal-opioids of interest extracted in anorganic phase may be transferred to an aqueous solution. In some cases,the organic solvent may be evaporated by heat and/or vacuum, and theresulting powder may be dissolved in an aqueous solution of suitable pH.In a further example, the BIAs of interest may be extracted from theorganic phase by addition of an aqueous solution at a suitable pH thatpromotes extraction of the nor-opioids or nal-opioids of interest intothe aqueous phase. The aqueous phase may then be removed by decantation,centrifugation, or another method.

The nor-opioid or nal-opioid-containing solution may be further treatedto remove metals, for example, by treating with a suitable chelatingagent. The nor-opioid or nal-opioid of interest-containing solution maybe further treated to remove other impurities, such as proteins and DNA,by precipitation. In one example, the nor-opioid or nal-opioid ofinterest-containing solution is treated with an appropriateprecipitation agent such as ethanol, methanol, acetone, or isopropanol.In an alternative example, DNA and protein may be removed by dialysis orby other methods of size exclusion that separate the smaller alkaloidsfrom contaminating biological macromolecules.

In further examples, the solution containing nor-opioids or nal-opioidsof interest may be extracted to high purity by continuous cross-flowfiltration using methods known in the art.

If the solution contains a mixture of nor-opioids or nal-opioids ofinterest, it may be subjected to acid-base treatment to yield individualnor-opioid or nal-opioid of interest species using methods known in theart. In this process, the pH of the aqueous solution is adjusted toprecipitate individual nor-opioids or nal-opioids.

For high purity, small-scale preparations, the nor-opioids ornal-opioids may be purified in a single step by liquid chromatography.

LCMS Method:

The BIA compounds of interest such as naloxone or naltrexone may beseparated using liquid chromatography, and detected and quantified usingmass spectrometry. Compound identity may be confirmed by characteristicelution time, mass-to-charge ratio (m/z) and fragmentation patterns(MS/MS). Quantitation may be performed by comparison of compound peakarea to a standard curve of a known reference standard compound.Additionally, BIAs of interest may be detected by alternative methodssuch as GC-MS, UV-vis spectroscopy, NMR, LC-NMR, LC-UV, TLC, andcapillary electrophoresis.

Purpald Assay Method

For high throughput screening of demethylation reactions a purpald assaymay be used. For example, demethylation catalyzed by 2-oxoglutaratedependent dioxygenases produces formaldehyde a as product as shown inthe generalized chemical equation:[substrate]+2-oxoglutarate+O₂⇄[product]+formaldehyde+succinate+CO₂.Purpald reagent in alkaline conditions undergoes a color change in thepresence of formaldehyde that can be quantified to concentrations as lowas 1 nM with a spectrophotometer at 510 nm.

Yeast-Derived Alkaloid APIs Versus Plant-Derived APIs

The clarified yeast culture medium (CYCM) may contain a plurality ofimpurities. The clarified yeast culture medium may be dehydrated byvacuum and/or heat to yield an alkaloid-rich powder. This product isanalogous to the concentrate of poppy straw (CPS), which is exportedfrom poppy-growing countries and purchased by Active PharmaceuticalIngredients (API) manufacturers. For the purposes of this invention, CPSis a representative example of any type of purified plant extract fromwhich the desired alkaloids product(s) may ultimately be furtherpurified. Table 9 and Table 10 highlight the impurities in these twoproducts that may be specific to either CYCM or CPS or may be present inboth. Accordingly, these nor-opioids or nal-opioids may be assessed forimpurities based on non-pigment impurities. By analyzing a product ofunknown origin for a subset of these impurities, a person of skill inthe art could determine whether the product originated from a yeast orplant production host.

API-grade pharmaceutical ingredients are highly purified molecules. Assuch, impurities that could indicate the plant- or yeast-origin of anAPI (such as those listed in Table 9 and Table 10) may not be present atthe API stage of the product. Indeed, many of the API products derivedfrom yeast strains of the present invention may be largelyindistinguishable from the traditional plant-derived APIs. In somecases, however, conventional alkaloid compounds may be subjected tochemical modification using chemical synthesis approaches, which mayshow up as chemical impurities in plant-based products that require suchchemical modifications. For example, chemical derivatization may oftenresult in a set of impurities related to the chemical synthesisprocesses. In certain situations, these modifications may be performedbiologically in the yeast production platform, thereby avoiding some ofthe impurities associated with chemical derivation from being present inthe yeast-derived product. In particular, these impurities from thechemical derivation product may be present in an API product that isproduced using chemical synthesis processes but may be absent from anAPI product that is produced using a yeast-derived product.Alternatively, if a yeast-derived product is mixed with achemically-derived product, the resulting impurities may be present butin a lesser amount than would be expected in an API that only orprimarily contains chemically-derived products. In this example, byanalyzing the API product for a subset of these impurities, a person ofskill in the art could determine whether the product originated from ayeast production host or the traditional chemical derivatization route.

Non-limiting examples of impurities that may be present inchemically-derivatized morphinan APIs but not in biosynthesized APIsinclude a codeine-O(6)-methyl ether impurity in API codeine;8,14-dihydroxy-7,8-dihydrocodeinone in API oxycodone; andtetrahydrothebaine in API hydrocodone. The codeine-O(6)-methyl ether maybe formed by chemical over-methylation of morphine. The8,14-dihydroxy-7,8-dihydrocodeinone in API oxycodone may be formed bychemical over-oxidation of thebaine. Additionally, thetetrahydrothebaine in API hydrocodone may be formed by chemicalover-reduction of thebaine.

However, in the case where the yeast-derived compound and theplant-derived compound are both subjected to chemical modificationthrough chemical synthesis approaches, the same impurities associatedwith the chemical synthesis process may be expected in the products. Insuch a situation, the starting material (e.g., CYCM or CPS) may beanalyzed as described above.

Host Cell Derived Nal-Opioids Vs Chemically Derived Nal-Opioids

Nal-opioids produced by chemical synthesis may contain a plurality ofimpurities. These impurities may arise from many different causes, forexample, unreacted starting materials, incomplete reactions, theformation of byproducts, persistence of intermediates, dimerization, ordegradation. An example of an unreacted starting material could beoxymorphone remaining in a preparation of naltrexone. An example of animpurity arising from an incomplete reaction could be3-O-Methylbuprenorphine resulting from the incomplete 3-O-demethylationof thebaine. Chemical modification can result in the addition or removalof functional groups at off-target sites. For example, the oxidation ofC10 to create 10-hydroxynaltrexone and 10-ketonaltrexone duringnaltrexone synthesis, or the removal of the 6-O-methyl group to give6-O-desmethylbuprenorphine during buprenorphine synthesis. Impuritiesmay arise from the persistence of reaction intermediates, for examplethe persistence of N-oxides like oxymorphone N-oxide formed during theN-demethylation process. Another source of impurities is dimerization,the conjugation of two opioid molecules, for example two buprenorphinemolecules (2,2′-bisbuprenorphine), two naltrexone molecules(2,2′-bisnaltrexone), or two naloxone molecules (2,2′-bisnaloxone).Impurities may arise from degradation of starting materials, reactionintermediates, or reaction products. The extreme physical conditionsused in chemical syntheses may make the presence of degradation morelikely. An example of an impurity that may arise from degradation isdehydrobuprenorphine produced by oxidizing conditions duringbuprenorphine synthesis.

Nal-opioids produced by enzyme catalysis in a host cell may containdifferent impurities than nal-opioids produced by chemical synthesis.Nal-opioids produced by enzyme catalysis in a host cell may containfewer impurities than nal-opioids produced by chemical synthesis.Nal-opioids produced by enzyme catalysis in a host cell may lack certainimpurities that are found in nal-opioids produced by chemical synthesis.In examples, key features of enzyme synthesis may include, (1) enzymestarget a specific substrate and residue with high fidelity; (2) enzymesperform reactions in the mild physiological conditions within the cellwhich do not compromise the stability of the molecules; and (3) enzymesare engineered to be efficient catalysts that drive reactions tocompletion.

Table 11 highlights some of the impurities that may be specific tochemically produced nal-opioids. Accordingly, nal-opioids may beassessed for impurities to determine the presence or absence of anyimpurity from Table 11. By analyzing a product of unknown origin for asubset of these impurities, a person of skill in the art could determinewhether the product originated from a chemical or enzymatic synthesis.

Methods of Engineering Host Cells

Also included are methods of engineering host cells for the purpose ofproducing nor-opioid and/or nal-opioid BIAs of interest. Inserting DNAinto host cells may be achieved using any convenient methods. Themethods are used to insert the heterologous coding sequences into theengineered host cells such that the host cells functionally express theenzymes and convert starting compounds of interest into productnor-opioid and/or nal-opioid BIAs of interest.

Any convenient promoters may be utilized in the subject engineered hostcells and methods. The promoters driving expression of the heterologouscoding sequences may be constitutive promoters or inducible promoters,provided that the promoters are active in the engineered host cells. Theheterologous coding sequences may be expressed from their nativepromoters, or non-native promoters may be used. Such promoters may below to high strength in the host in which they are used. Promoters maybe regulated or constitutive. In certain embodiments, promoters that arenot glucose repressed, or repressed only mildly by the presence ofglucose in the culture medium, are used. Promoters of interest includebut are not limited to, promoters of glycolytic genes such as thepromoter of the B. subtilis tsr gene (encoding the promoter region ofthe fructose bisphosphate aldolase gene) or the promoter from yeast S.cerevisiae gene coding for glyceraldehyde 3-phosphate dehydrogenase(GPD, GAPDH, or TDH3), the ADH1 promoter of baker's yeast, thephosphate-starvation induced promoters such as the PHO5 promoter ofyeast, the alkaline phosphatase promoter from B. licheniformis, yeastinducible promoters such as Gal1-10, Gal1, GalL, GalS, repressiblepromoter Met25, tetO, and constitutive promoters such as glyceraldehyde3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter(ADH), translation-elongation factor-1-α promoter (TEF), cytochromec-oxidase promoter (CYC1), MRP7 promoter, etc. Autonomously replicatingyeast expression vectors containing promoters inducible by hormones suchas glucocorticoids, steroids, and thyroid hormones may also be used andinclude, but are not limited to, the glucorticoid responsive element(GRE) and thyroid hormone responsive element (TRE). These and otherexamples are described U.S. Pat. No. 7,045,290, which is incorporated byreference, including the references cited therein. Additional vectorscontaining constitutive or inducible promoters such as a factor, alcoholoxidase, and PGH may be used. Additionally any promoter/enhancercombination (as per the Eukaryotic Promoter Data Base EPDB) could alsobe used to drive expression of genes. Any convenient appropriatepromoters may be selected for the host cell, examples of promoters thatcould be used in an E. coli cell include T7, lac and tetO promoters. Onemay also use promoter selection to optimize transcript, and hence,enzyme levels to maximize production while minimizing energy resources.

Any convenient vectors may be utilized in the subject engineered hostcells and methods. Vectors of interest include vectors for use in yeastand other cells. The types of yeast vectors may be broken up into 4general categories: integrative vectors (YIp), autonomously replicatinghigh copy-number vectors (YEp or 2μ plasmids), autonomously replicatinglow copy-number vectors (YCp or centromeric plasmids) and vectors forcloning large fragments (YACs). Vector DNA is introduced intoprokaryotic or eukaryotic cells via any convenient transformation ortransfection techniques. DNA of another source (e.g. PCR-generateddouble stranded DNA product, or synthesized double stranded or singlestranded oligonucleotides) may be used to engineer the yeast byintegration into the genome. Any single transformation event may includeone or several nucleic acids (vectors, double stranded or singlestranded DNA fragments) to genetically modify the host cell.

Utility

The engineered host cells and methods disclosed herein, e.g., asdescribed above, find use in a variety of applications. Applications ofinterest include, but are not limited to: research applications andtherapeutic applications. Methods disclosed herein find use in a varietyof different applications including any convenient application where theproduction of nor-opioid and/or nal-opioid BIAs of interest.

The subject engineered host cells and methods find use in a variety oftherapeutic applications. Therapeutic applications of interest includethose applications in which the preparation of pharmaceutical productsthat include nor-opioids or nal-opioids is of interest. The engineeredhost cells described herein produce nor-opioids or nal-opioids ofinterest and enzymes of interest. Reticuline is a major branch pointintermediate of interest in the synthesis of BIAs including engineeringefforts to produce end products such as opioid products. The subjecthost cells may be utilized to produce nor-opioids or nal-opioids ofinterest from simple and inexpensive starting materials that may finduse in the production of BIAs of interest, including reticuline, and BIAend products, such as nor-opioids or nal-opioids. As such, the subjecthost cells find use in the supply of therapeutically active nor-opioidsor nal-opioids of interest.

In some instances, the engineered host cells and methods find use in theproduction of commercial scale amounts of nor-opioids or nal-opioidsthereof where chemical synthesis of these compounds is low yielding andnot a viable means for large-scale production. In certain cases, thehost cells and methods are utilized in a fermentation facility thatwould include bioreactors (fermenters) of e.g., 5,000-200,000 litercapacity allowing for rapid production of nor-opioids or nal-opioids ofinterest thereof for therapeutic products. Such applications may includethe industrial-scale production of nor-opioids or nal-opioids ofinterest from fermentable carbon sources such as cellulose, starch, andfree sugars.

The subject engineered host cells and methods find use in a variety ofresearch applications. The subject host cells and methods may be used toanalyze the effects of a variety of enzymes on the biosynthetic pathwaysof a variety of nor-opioid and/or nal-opioid BIAs of interest. Inaddition, the engineered host cells may be engineered to producenor-opioid and/or nal-opioid BIAs of interest that find use in testingfor bioactivity of interest in as yet unproven therapeutic functions. Insome cases, the engineering of host cells to include a variety ofheterologous coding sequences that encode for a variety of enzymeselucidates the high yielding biosynthetic pathways towards nor-opioidand/or nal-opioid BIAs of interest. In certain cases, researchapplications include the production of nor-opioid and/or nal-opioid BIAsof interest for therapeutic molecules of interest that may then befurther chemically modified or derivatized to desired products or forscreening for increased therapeutic activities of interest. In someinstances, host cell strains are used to screen for enzyme activitiesthat are of interest in such pathways, which may lead to enzymediscovery via conversion of nor-opioid or nal-opioid metabolitesproduced in these strains.

The subject engineered host cells and methods may be used as aproduction platform for plant specialized metabolites. The subject hostcells and methods may be used as a platform for drug library developmentas well as plant enzyme discovery. For example, the subject engineeredhost cells and methods may find use in the development of naturalproduct based drug libraries by taking yeast strains producinginteresting scaffold molecules, such as norcodeine, or northebaine, andfurther functionalizing the compound structure through combinatorialbiosynthesis or by chemical means. By producing drug libraries in thisway, any potential drug hits are already associated with a productionhost that is amenable to large-scale culture and production. As anotherexample, these subject engineered host cells and methods may find use inplant enzyme discovery. The subject host cells provide a cleanbackground of defined metabolites to express plant EST libraries toidentify new enzyme activities. The subject host cells and methodsprovide expression methods and culture conditions for the functionalexpression and increased activity of plant enzymes in yeast.

Kits and Systems

Some aspects of the invention further include kits and systems, wherethe kits and systems may include one or more components employed inmethods disclosed herein, e.g., engineered host cells, startingcompounds, heterologous coding sequences, vectors, culture medium, etc.,as described herein. In some embodiments, the subject kit includes anengineered host cell (e.g., as described herein), and one or morecomponents selected from the following: starting compounds, aheterologous coding sequence and/or a vector including the same,vectors, growth feedstock, components suitable for use in expressionsystems (e.g., cells, cloning vectors, multiple cloning sites (MCS),bi-directional promoters, an internal ribosome entry site (IRES), etc.),and a culture medium.

Any of the components described herein may be provided in the kits,e.g., host cells including one or more modifications, startingcompounds, culture medium, etc. A variety of components suitable for usein making and using heterologous coding sequences, cloning vectors andexpression systems may find use in the subject kits. Kits may alsoinclude tubes, buffers, etc., and instructions for use. The variousreagent components of the kits may be present in separate containers, orsome or all of them may be pre-combined into a reagent mixture in asingle container, as desired.

Also provided are systems for producing nor-opioid and/or nal-opioidBIAs of interest, where the systems may include engineered host cellsincluding one or more modifications (e.g., as described herein),starting compounds, culture medium, a fermenter and fermentationequipment, e.g., an apparatus suitable for maintaining growth conditionsfor the host cells, sampling and monitoring equipment and components,and the like. A variety of components suitable for use in large scalefermentation of yeast cells may find use in the subject systems.

In some cases, the system includes components for the large scalefermentation of engineered host cells, and the monitoring andpurification of nor-opioid or nal-opioid compounds produced by thefermented host cells. In certain embodiments, one or more startingcompounds (e.g., as described herein) are added to the system, underconditions by which the engineered host cells in the fermenter produceone or more desired nor-opioid or nal-opioid products of interest. Insome instances, the host cells produce a nor-opioid or nal-opioid ofinterest (e.g., as described herein). In certain cases, the nor-opioidor nal-opioid products of interest are opioid antagonists, such asnaloxone, naltrexone, nalmefene, or nalorphine. In certain cases, thenor-opioid or nal-opioid products of interest are opioid antagonistssuch as naltrindole or norbinaltorphimine. In some examples, thenor-opioid or nal-opioid products of interest are partial agonists suchas buprenorphine.

In some cases, the system includes processes for monitoring and oranalyzing one or more nor-opioid and/or nal-opioid BIAs of interestcompounds produced by the subject host cells. For example, a LC-MSanalysis system as described herein, a chromatography system, or anyconvenient system where the sample may be analyzed and compared to astandard, e.g., as described herein. The fermentation medium may bemonitored at any convenient times before and during fermentation bysampling and analysis. When the conversion of starting compounds tonor-opioid or nal-opioid products of interest is complete, thefermentation may be halted and purification of the nor-opioid ornal-opioid products may be done. As such, in some cases, the subjectsystem includes a purification component suitable for purifying thenor-opioid or nal-opioid products of interest from the host cell mediuminto which it is produced. The purification component may include anyconvenient means that may be used to purify the nor-opioid or nal-opioidproducts of interest produced by fermentation, including but not limitedto, silica chromatography, reverse-phase chromatography, ion exchangechromatography, HIC chromatography, size exclusion chromatography,liquid extraction, and pH extraction methods. In some cases, the subjectsystem provides for the production and isolation of enzyme and/ornor-opioid or nal-opioid fermentation products of interest following theinput of one or more starting compounds to the system.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.), but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Discussion of Enzyme List

The host cells may be engineered to include one or more modifications(such as two or more, three or more, four or more, five or more, or evenmore modifications) that provide for the production of nor-opioid and/ornal-opioid BIAs of interest. Table 2 provides a list of exemplary genesthat may be acted upon by one or more modifications so as to provide forthe production of nor-opioid and/or nal-opioid BIAs of interest and/orenzymes of interest in an engineered host cell.

Modifications of genes as provided in Table 2 may be used to producenor-opioid and/or nal-opioid BIAs of interest from engineered host cellsthat are supplied with a medium containing the minimal nutrientsrequired for growth. This minimal medium may contain a carbon source, anitrogen source, amino acids, vitamins, and salts. For example,modifications of genes as provided in Table 2 may be used to producenor-opioid and/or nal-opioid BIAs of interest from engineered host cellsthat are fed sugar. Additionally, modifications of one or more genes asprovided in Table 2 may be used to augment the biosynthetic processes ofhost cells that may be engineered for drug production.

Additionally, the use of these modifications to provide for theproduction of nor-opioid and/or nal-opioid BIAs of interest and/orenzymes of interest in engineered host cells is not readily apparentfrom the mere identification of enzymes that may be produced by thegenes. In particular, synthetic pathways that have been reconstructed inhost cells, such as yeast cells, as described herein comprise a varietyof enzymes that do not act together in nature within a single organism.Additionally, some of the enzymes discussed herein do not act fornor-opioid and/or nal-opioid BIA biosynthesis in their natural context.Further, some of the enzymes described herein are not evolved tofunction in particular host cells, such as yeast cells, and are notevolved to function together. Further, some of the nor-opioids ornal-opioids produced do not occur naturally. In these cases, it wouldnot be obvious that the enzymes would exhibit sufficient activity in thecontext of the synthetic nor-opioid and/or nal-opioid pathway in a hostcell, such as yeast, to have sufficient flux through the pathway toproduce downstream nor-opioid or nal-opioid end products.

For example, plant enzymes are often difficult to functionally expressin heterologous microbial hosts, such as yeast. In many cases theenzymes may be misfolded, not correctly localized within the host cell,and/or incorrectly processed. The differences in protein translation andprocessing between yeast and plants can lead to these enzymes exhibitingsubstantially reduced to no detectable activities in the yeast host.These challenges arise commonly for endomembrane localized enzymes, suchas cytochrome P450s, which are strongly represented in the BIA pathwayswhich produce precursors for nor-opioids or nal-opioids. Even reducedenzyme activities may pose a substantial challenge to engineering yeastto produce complex BIAs, which requires sufficient activity at each stepto ensure high-level accumulation of the desired BIA products.

Additionally, there are endogenous enzymes/pathways in some host cells,such as yeast, that may act on many of the early precursors in the BIApathway (i.e., intermediates from tyrosine to norcoclaurine), and thusit may not be readily apparent that there would be sufficient fluxthrough the heterologous pathway to achieve substantial BIA productiongiven these competing endogenous pathways. For example, the Erlichpathway (Hazelwood, et al. 2008. Appl. Environ. Microbiol. 74: 2259-66;Larroy, et al. 2003. Chem. Biol. Interact. 143-144: 229-38; Larroy, etal. 2002. Eur. J. Biochem. 269: 5738-45) in yeast is the main endogenouspathway that would act to convert many of the intermediates in the earlyBIA pathway to undesired products and divert flux from the syntheticpathway.

Further, many of the enzymes as discussed herein, and as provided inTable 2, may function under very specific regulation strategies,including spatial regulation, in the native plant hosts, which may belost upon transfer to the heterologous yeast host. In addition, plantspresent very different biochemical environments than yeast cells underwhich the enzymes are evolved to function, including pH, redox state,and substrate, cosubstrate, coenzyme, and cofactor availabilities. Giventhe differences in biochemical environments and regulatory strategiesbetween the native hosts and the heterologous yeast hosts, it is notobvious that the enzymes would exhibit substantial activities when inthe context of the yeast environment and further not obvious that theywould work together to direct simple precursors such as sugar to complexBIA compounds. Maintaining the activities of the enzymes in the yeasthost is particularly important as many of the pathways have manyreaction steps (>10), such that if these steps are not efficient thenone would not expect accumulation of desired downstream products.

In addition, in the native plant hosts, the associated metabolites inthese pathways may be localized across different cell and tissue types.In several examples, there are cell types that may be specialized forbiosynthesis and cell types that may be synthesized for metaboliteaccumulation. This type of cell specialization may be lost whenexpressing the pathways within a heterologous yeast host, and may playan important role in controlling the toxicity of these metabolites onthe cells. Thus, it is not obvious that yeast could be successfullyengineered to biosynthesize and accumulate these metabolites withoutbeing harmed by the toxicity of these compounds.

As one example, in the native plant hosts, the enzyme BBE is reported tohave dynamic subcellular localization. In particular, the enzyme BBEinitially starts in the ER and then is sorted to the vacuole (Bird andFacchini. 2001. Planta. 213: 888-97). It has been suggested that theER-association of BBE in plants (Alcantara, et al. 2005. Plant Physiol.138: 173-83) provides the optimal basic pH (pH ˜8.8) for BBE activity(Ziegler and Facchini. 2008. Annu. Rev. Plant Biol. 59: 735-69). Asanother example, there is evidence that sanguinarine biosynthesis occursin specialized vesicles within plant cells (Amann, et al. 1986. Planta.167: 310-20), but only some of the intermediates accumulate in thevesicles. This may occur so as to sequester them from other enzymeactivities and/or toxic effects.

As another example, the biosynthetic enzymes in the morphinan pathwaybranch are all localized to the phloem, which is part of the vasculartissue in plants. In the phloem, the pathway enzymes may be furtherdivided between two cell types: the sieve elements common to all plants,and the laticifer which is a specialized cell type present only incertain plants which make specialized secondary metabolites. Theupstream enzymes (i.e., from NCS through to SalAT) are predominantly inthe sieve elements, and the downstream enzymes (i.e., T6ODM, COR, CODM)are mostly in the laticifer (Onoyovwe, et al. 2013. Plant Cell. 25:4110-22). Additionally, it was discovered that the final steps in thenoscapine biosynthetic pathway take place in the laticifer (Chen andFacchini. 2014. Plant J. 77: 173-84). This compartmentalization isthought to be highly important for regulating biosynthesis by isolatingor trafficking intermediates, providing optimal pH, enhancing supply ofcofactors, although the nature of the poppy laticifer microenvironmentis still under investigation (Ziegler and Facchini. 2008. Annu. Rev.Plant Biol. 59: 735-69). Further, it is predicted that several of theenzymes may function as multi-enzyme complexes or metabolic channelscommon to plant secondary metabolism (Kempe, et al. 2009.Phytochemistry. 70: 579-89; Allen, et al. 2004. Nat. Biotechnol. 22:1559-66). When biosynthetic enzymes are combined from different hostsand/or expressed recombinantly in a heterologous yeast cell it is notclear that these complexes or channels will form as they would in thenative host. In an additional example, in Coptis japonica, berberine isbiosynthesized in root tissues and then accumulated within the rhizomevia the action of specialized ATP-binding cassette transport proteins(Shitan, et al. 2013. Phytochemistry. 91: 109-16). In opium poppy,morphinan alkaloids are accumulated within the latex (cytoplasm oflaticifer cells) (Martin, et al. 1967. Biochemistry. 6: 2355-63).

Further, even without these considerations, it is also the case that theplant enzymes for several of the steps in the pathways described hereinhave not yet been characterized. For example, the conversion of tyrosineto the early benzylisoquinoline alkaloid scaffold norcoclaurine has notyet been characterized. Thus, for several of the steps in the pathwaysdescribed herein, alternative biosynthetic scheme were produced bybringing together enzyme activities that do not normally occur togetherin nature for the biosynthesis of BIAs or identifying new enzymeactivities from genome sequence information to use in the reconstructedpathways.

For example, the two-step conversion of tyrosine to dopamine may beachieved by combining at least 5 mammalian enzymes and 1 bacterialenzyme, which do not naturally occur together and were not evolved tofunction in the context of this pathway or with plant enzymes. In theseinstances, it may not be obvious to utilize these enzymes for thebiosynthesis of compounds they were not evolved for in nature and thatthey would function effectively in the context of a heterologousmicrobial host and this pathway.

Examples of the genes that are the object of modifications so as toproduce nor-opioid and/or nal-opioid BIAs of interest and/or enzymes ofinterest are discussed below. Additionally, the genes are discussed inthe context of a series of Figures that illustrate pathways that areused in generating BIAs and nor-opioid and/or nal-opioid BIAs ofinterest and/or enzymes of interest.

[TLK1] In some examples, the engineered host cell may modify theexpression of the enzyme transketolase. Transketolase is encoded by theTKL1 gene. In examples, transketolase catalyzes the reaction offructose-6-phosphate+glyceraldehyde-3-phosphate↔xylulose-5-phosphate+erythrose-4-phosphate,as referenced in FIG. 2. An engineered host cell may be modified toinclude constitutive overexpression of the TKL1 gene in the engineeredhost cell. Additionally or alternatively, the engineered host cell maybe modified to synthetically regulate the expression of the TKL1 gene inthe engineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of theTKL1 gene. Additionally or alternatively, the engineered host cell maybe modified to incorporate the introduction of a strong promoter elementfor the overexpression of the TKL1 gene within the engineered host cell.The TKL1 gene may be derived from Saccharomyces cerevisiae or anotherspecies. In some examples, the TKL1 gene may be 100% similar to thenaturally occurring gene.

[ZWF1] In some examples, the engineered host cell may modify theexpression of the enzyme glucose-6-phosphate dehydrogenase.Glucose-6-phosphate dehydrogenase is encoded by the ZWF1 gene. Inexamples, glucose-6-phosphate dehydrogenase catalyzes the reaction ofglucose-6-phosphate→6-phosphogluconolactone, as referenced in FIG. 2. Anengineered host cell may be modified to delete the coding region of theZWF1 gene in the engineered host cell. Alternatively, the engineeredhost cell may be modified to disable the functionality of the ZWF1 gene,such as by introducing an inactivating mutation.

[ARO4] In some examples, the engineered host cell may modify theexpression of the enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate(DAHP) synthase. DAHP synthase is encoded by the ARO4 gene. In examples,DAHP synthase catalyzes the reaction oferythrose-4-phosphate+phosphoenolpyruvic acid→DAHP, as referenced inFIG. 2. An engineered host cell may modify the ARO4 gene to incorporateone or more feedback inhibition alleviating mutations. In particular, afeedback inhibition alleviating mutation (e.g., ARO4^(FBR)) may beincorporated as a directed mutation to a native ARO4 gene at theoriginal locus; as an additional copy introduced as a geneticintegration at a separate locus; or as an additional copy on an episomalvector such as a 2-μm or centromeric plasmid. The identifier “FBR” inthe mutation ARO4^(FBR) refers to feedback resistant mutants andmutations. The feedback inhibited copy of the DAHP synthase enzyme maybe under a native yeast transcriptional regulation, such as when theengineered host cell is a yeast cell. Alternatively, the feedbackinhibited copy of the DAHP synthase enzyme may be introduced to theengineered host cell with engineered constitutive or dynamic regulationof protein expression by placing it under the control of a syntheticpromoter. In some cases, the ARO4 gene may be derived from Saccharomycescerevisiae. In some cases, the ARO4 gene may be 100% similar to thenaturally occurring gene. Examples of modifications to the ARO4 geneinclude a feedback inhibition resistant mutation, K229L, or Q166K.

[ARO7] In some examples, the engineered host cell may modify theexpression of the enzyme chorismate mutase. Chorismate mutase is encodedby the ARO7 gene. In examples, chorismate mutase catalyzes the reactionof chorismate→prephenate, as referenced in FIG. 2. An engineered hostcell may modify the ARO7 gene to incorporate one or more feedbackinhibition alleviating mutations. In particular, a feedback inhibitionalleviating mutation (e.g., ARO7^(FBR)) may be incorporated as adirected mutation to a native ARO7 gene at the original locus; as anadditional copy introduced as a genetic integration at a separate locus;or as an additional copy on an episomal vector such as a 2-μm orcentromeric plasmid. The identifier “FBR” in the mutation ARO7^(FBR)refers to feedback resistant mutants and mutations. The feedbackinhibited copy of the chorismate mutase enzyme may be under a nativeyeast transcriptional regulation, such as when the engineered host cellis a yeast cell. Alternatively, the feedback inhibited copy of thechorismate mutase enzyme may be introduced to the engineered host cellwith engineered constitutive or dynamic regulation of protein expressionby placing it under the control of a synthetic promoter. In some cases,the ARO7 gene may be derived from Saccharomyces cerevisiae. In somecases, the ARO7 gene may be 100% similar to the naturally occurringgene. Examples of modifications to the ARO7 gene include a feedbackinhibition resistant mutation or T226I.

[ARO10] In some examples, the engineered host cell may modify theexpression of the enzyme phenylpyruvate decarboxylase. Phenylpyruvatedecarboxylase is encoded by the ARO10 gene. In examples, phenylpyruvatedecarboxylase catalyzes the reaction ofhydroxyphenylpyruvate→4-hydroxyphenylacetate (4HPA), as referenced inFIG. 2. An engineered host cell may be modified to include constitutiveoverexpression of the ARO10 gene in the engineered host cell.Additionally or alternatively, the engineered host cell may be modifiedto synthetically regulate the expression of the ARO10 gene in theengineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of theARO10 gene. Additionally or alternatively, the engineered host cell maybe modified to incorporate the introduction of a strong promoter elementfor the overexpression of the ARO10 gene within the engineered hostcell. The ARO10 gene may be derived from Saccharomyces cerevisiae oranother species. In some examples, the ARO10 gene may be 100% similar tothe naturally occurring gene.

[ADH2-7, SFA1] In some examples, the engineered host cell may modify theexpression of alcohol dehydrogenase enzymes. Alcohol dehydrogenaseenzymes may be encoded by one or more of the ADH2, ADH3, ADH4, ADH5,ADH6, ADH7, and SFA1 genes. In examples, alcohol dehydrogenase catalyzesthe reaction of 4HPA→tyrosol. An engineered host cell may be modified todelete the coding region of one or more of the ADH2, ADH3, ADH4, ADH5,ADH6, ADH7, and SFA1 genes in the engineered host cell. Alternatively,the engineered host cell may be modified to disable the functionality ofone or more of the ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, and SFA1 genes,such as by introducing an inactivating mutation.

[ALD2-6] In some examples, the engineered host cell may modify theexpression of aldehyde oxidase enzymes. Aldehyde oxidase enzymes may beencoded by one or more of the ALD2, ALD3, ALD4, ALD5, and ALD6 genes. Inexamples, aldehyde oxidase catalyzes the reaction of4HPA→hydroxyphenylacetic acid. An engineered host cell may be modifiedto delete the coding region of one or more of the ALD2, ALD3, ALD4,ALD5, and ALD6 genes in the engineered host cell. Alternatively, theengineered host cell may be modified to disable the functionality of oneor more of the ALD2, ALD3, ALD4, ALD5, and ALD6 genes, such as byintroducing an inactivating mutation.

[ARO9] In some examples, the engineered host cell may modify theexpression of the enzyme aromatic aminotransferase. Aromaticaminotransferase is encoded by the ARO9 gene. In examples, aromaticaminotransferase catalyzes the reaction ofhydroxyphenylpyruvate+glutamate→tyrosine+alpha-ketogluterate, asreferenced in FIG. 2. An engineered host cell may be modified to includeconstitutive overexpression of the ARO9 gene in the engineered hostcell. Additionally or alternatively, the engineered host cell may bemodified to synthetically regulate the expression of the ARO9 gene inthe engineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of theARO9 gene. Additionally or alternatively, the engineered host cell maybe modified to incorporate the introduction of a strong promoter elementfor the overexpression of the ARO9 gene within the engineered host cell.The ARO9 gene may be derived from Saccharomyces cerevisiae or anotherspecies. In some examples, the ARO9 gene may be 100% similar to thenaturally occurring gene.

[TYR] In some examples, the engineered host cell may modify theexpression of the enzyme tyrosinase. Tyrosinase is encoded by the TYRgene. In examples, tyrosinase catalyzes the reaction of tyrosine→L-DOPA,as referenced in FIG. 2. In other examples, tyrosinase catalyzes thereaction of L-DOPA→dopaquinone. An engineered host cell may be modifiedto include constitutive expression of the TYR gene in the engineeredhost cell. Additionally or alternatively, the engineered host cell maybe modified to synthetically regulate the expression of the TYR gene inthe engineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of the TYRgene. Additionally or alternatively, the engineered host cell may bemodified to incorporate the introduction of a strong promoter elementfor the overexpression of the TYR gene within the engineered host cell.The TYR gene may be derived from Ralstonia solanacearum, Agaricusbisporus, or another species. In some examples, the TYR gene may be 100%similar to the naturally occurring gene.

[TyrH] In some examples, the engineered host cell may modify theexpression of the enzyme tyrosine hydroxylase. Tyrosine hydroxylase isencoded by the TyrH gene. In examples, tyrosine hydroxylase catalyzesthe reaction of tyrosine→L-DOPA, as referenced in FIGS. 2 and 5. Anengineered host cell may be modified to include constitutive expressionof the TyrH gene in the engineered host cell. Additionally oralternatively, the engineered host cell may be modified to syntheticallyregulate the expression of the TyrH gene in the engineered host cell. Inexamples, the engineered host cell may be modified to incorporate acopy, copies, or additional copies, of the TyrH gene. Additionally oralternatively, the engineered host cell may be modified to incorporatethe introduction of a strong promoter element for the overexpression ofthe TyrH gene within the engineered host cell. The TyrH gene may bederived from Homo sapiens, Rattus norvegicus, Mus musculus, or anotherspecies. In some examples, the TyrH gene may be 100% similar to thenaturally occurring gene.

[DODC] In some examples, the engineered host cell may modify theexpression of the enzyme L-DOPA decarboxylase. L-DOPA decarboxylase isencoded by the DODC gene. In examples, L-DOPA decarboxylase catalyzesthe reaction of L-DOPA→dopamine, as referenced in FIGS. 2 and 5. Anengineered host cell may be modified to include constitutive expressionof the DODC gene in the engineered host cell. Additionally oralternatively, the engineered host cell may be modified to syntheticallyregulate the expression of the DODC gene in the engineered host cell. Inexamples, the engineered host cell may be modified to incorporate acopy, copies, or additional copies, of the DODC gene. Additionally oralternatively, the engineered host cell may be modified to incorporatethe introduction of a strong promoter element for the overexpression ofthe DODC gene within the engineered host cell. The DODC gene may bederived from Pseudomonas putida, Rattus norvegicus, or another species.In some examples, the DODC gene may be 100% similar to the naturallyoccurring gene.

[TYDC] In some examples, the engineered host cell may modify theexpression of the enzyme tyrosine/DOPA decarboxylase. Tyrosine/DOPAdecarboxylase is encoded by the TYDC gene. In examples, tyrosine/DOPAdecarboxylase catalyzes the reaction of L-DOPA→dopamine, as referencedin FIG. 2. An engineered host cell may be modified to includeconstitutive expression of the TYDC gene in the engineered host cell.Additionally or alternatively, the engineered host cell may be modifiedto synthetically regulate the expression of the TYDC gene in theengineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of theTYDC gene. Additionally or alternatively, the engineered host cell maybe modified to incorporate the introduction of a strong promoter elementfor the overexpression of the TYDC gene within the engineered host cell.The TYDC gene may be derived from Papaver somniferum or another species.In some examples, the TYDC gene may be 100% similar to the naturallyoccurring gene.

[MAO] In some examples, the engineered host cell may modify theexpression of the enzyme monoamine oxidase. Monoamine oxidase is encodedby the MAO gene. In examples, monoamine oxidase catalyzes the reactionof dopamine→3,4-DHPA, as referenced in FIG. 2. An engineered host cellmay be modified to include constitutive expression of the MAO gene inthe engineered host cell. Additionally or alternatively, the engineeredhost cell may be modified to synthetically regulate the expression ofthe MAO gene in the engineered host cell. In examples, the engineeredhost cell may be modified to incorporate a copy, copies, or additionalcopies, of the MAO gene. Additionally or alternatively, the engineeredhost cell may be modified to incorporate the introduction of a strongpromoter element for the overexpression of the MAO gene within theengineered host cell. In some cases, the MAO gene may be codon optimizedfor expression in Saccharomyces cerevisiae. The MAO gene may be derivedfrom Escherichia coli, Homo sapiens, Micrococcus luteus, or anotherspecies. In some examples, the MAO gene may be 77% similar to thenaturally occurring gene.

[NCS] In some examples, the engineered host cell may modify theexpression of the enzyme norcoclaurine synthase. Norcoclaurine synthaseis encoded by the NCS gene. In examples, norcoclaurine synthasecatalyzes the reaction of 4HPA+dopamine→(S)-norcoclaurine, as referencedin FIG. 5. In particular, FIG. 5 illustrates a biosynthetic scheme forconversion of L-tyrosine to reticuline via norcoclaurine, in accordancewith embodiments of the invention. FIG. 5 provides the use of theenzymes TyrH, tyrosine hydroxylase; DODC, DOPA decarboxylase; NCS,norcoclaurine synthase, as discussed herein; 6OMT,6-O-methyltransferase; CNMT, coclaurine N-methyltransferase; CYP80B1,cytochrome P450 80B1; CPR, cytochrome P450 NADPH reductase; 4′OMT,3′hydroxy-N-methylcoclaurine 4′-O-methyltransferase. L-DOPA,L-3,4-dihydroxyphenylalanine; and 4-HPA, 4-hydroxyphenylacetylaldehyde.Of the enzymes that are illustrated in FIG. 5, 4-HPA and L-tyrosine arenaturally synthesized in yeast. All other metabolites shown are notnaturally produced in yeast. Additionally, although TyrH is depicted ascatalyzing the conversion of L-tyrosine to L-DOPA, other enzymes mayalso be used to perform this step as described in the specification. Forexample, tyrosinases may also be used to perform the conversion ofL-tyrosine to L-DOPA. In addition, other enzymes such as cytochrome P450oxidases may also be used to perform the conversion of L-tyrosine toL-DOPA. Such enzymes may exhibit oxidase activity on related BIAprecursor compounds including L-DOPA and L-tyrosine.

Additionally, norcoclaurine synthase catalyzes the reaction of3,4-DHPA+dopamine→(S)-norlaudanosoline, as referenced in FIG. 6. Inparticular, FIG. 6 illustrates a biosynthetic scheme for conversion ofL-tyrosine to reticuline via norlaudanosoline, in accordance withembodiments of the invention. FIG. 6 provides the use of the enzymesTyrH, tyrosine hydroxylase; DODC, DOPA decarboxylase; maoA, monoamineoxidase; NCS, norcoclaurine synthase; 6OMT, 6-O-methyltransferase; CNMT,coclaurine N-methyltransferase; 4′OMT, 3′hydroxy-N-methylcoclaurine4′-O-methyltransferase. L-DOPA, L-3,4-dihydroxyphenylalanine; and3,4-DHPA, 3,4-dihydroxyphenylacetaldehyde. Of the enzymes that areillustrated in FIG. 6, L-tyrosine is naturally synthesized in yeast.Other metabolites that are shown in FIG. 6 are not naturally produced inyeast.

An engineered host cell may be modified to include constitutiveexpression of the NCS gene in the engineered host cell. Additionally oralternatively, the engineered host cell may be modified to syntheticallyregulate the expression of the NCS gene in the engineered host cell. Inexamples, the engineered host cell may be modified to incorporate acopy, copies, or additional copies, of the NCS gene. Additionally oralternatively, the engineered host cell may be modified to incorporatethe introduction of a strong promoter element for the overexpression ofthe NCS gene within the engineered host cell. Additionally, thenorcoclaurine synthase may have an N-terminal truncation. In some cases,the NCS gene may be codon optimized for expression in Saccharomycescerevisiae. The NCS gene may be derived from Coptis japonica, Papaversomniferum, Papver bracteatum, Thalicitum flavum, Corydalis saxicola, oranother species. In some examples, the NCS gene may be 80% similar tothe naturally occurring gene.

[6OMT] In some examples, the engineered host cell may modify theexpression of the enzyme norcoclaurine 6-O-methyltransferase.Norcoclaurine 6-O-methyltransferase is encoded by the 6OMT gene. In someexamples, norcoclaurine 6-O-methyltransferase catalyzes the reaction ofnorcoclaurine→coclaurine, as referenced in FIG. 5. In other examples,norcoclaurine 6-O-methyltransferase catalyzes the reaction ofnorlaudanosoline→3′hydroxycoclaurine, as well as other reactionsdetailed herein, such as those provided in FIG. 6. Additionally, theengineered host cell may be modified to include constitutive expressionof the 6OMT gene in the engineered host cell. Additionally oralternatively, the engineered host cell may be modified to syntheticallyregulate the expression of the 6OMT gene in the engineered host cell. Inexamples, the engineered host cell may be modified to incorporate acopy, copies, or additional copies, of the 6OMT gene. Additionally oralternatively, the engineered host cell may be modified to incorporatethe introduction of a strong promoter element for the overexpression ofthe 6OMT gene within the engineered host cell. The 6OMT gene may bederived from P. somniferum, T. flavum, Coptis japonica, or anotherspecies. In some examples, the 6OMT gene may be 100% similar to thenaturally occurring gene.

[CNMT] In some examples, the engineered host cell may modify theexpression of the enzyme coclaurine-N-methyltransferase.Coclaurine-N-methyltransferase is encoded by the CNMT gene. In someexamples, coclaurine-N-methyltransferase catalyzes the reaction ofcoclaurine→N-methylcoclaurine, as referenced in FIG. 5. In otherexamples, the coclaurine-N-methyltransferase enzyme may catalyze thereaction of 3′hydroxycoclaurine→3′hydroxy-N-methylcoclaurine. In otherexamples, coclaurine-N-methyltransferase may catalyze the reaction ofnoroxymorphone→naloxone, as referenced in FIG. 26. In other examples,coclaurine-N-methyltransferase may catalyze other reactions detailedherein, such as those provided in FIG. 6. Additionally, the engineeredhost cell may be modified to include constitutive expression of the CNMTgene in the engineered host cell. Additionally or alternatively, theengineered host cell may be modified to synthetically regulate theexpression of the CNMT gene in the engineered host cell. In examples,the engineered host cell may be modified to incorporate a copy, copies,or additional copies, of the CNMT gene. Additionally or alternatively,the engineered host cell may be modified to incorporate the introductionof a strong promoter element for the overexpression of the CNMT genewithin the engineered host cell. The CNMT gene may be derived from P.somniferum, T. flavum, Coptis japonica, or another species. In someexamples, the CNMT gene may be 100% similar to the naturally occurringgene.

[4′OMT] In some examples, the engineered host cell may modify theexpression of the enzyme 4′-O-methyltransferase. 4′-O-methyltransferaseis encoded by the 4′OMT gene. In some examples, 4′-O-methyltransferasecatalyzes the reaction of 3′-hydroxy-N-methylcoclaurine→reticuline, asreferenced in FIG. 5. In other examples, 4′-O-methyltransferasecatalyzes other reactions detailed herein, such as those provided inFIG. 6. Additionally, the engineered host cell may be modified toinclude constitutive expression of the 4′OMT gene in the engineered hostcell. Additionally or alternatively, the engineered host cell may bemodified to synthetically regulate the expression of the 4′OMT gene inthe engineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of the4′OMT gene. Additionally or alternatively, the engineered host cell maybe modified to incorporate the introduction of a strong promoter elementfor the overexpression of the 4′OMT gene within the engineered hostcell. The 4′OMT gene may be derived from P. somniferum, T. flavum,Coptis japonica, or another species. In some examples, the 4′OMT genemay be 100% similar to the naturally occurring gene.

[CYP80B1] In some examples, the engineered host cell may modify theexpression of the enzyme cytochrome P450 80B1. Cytochrome P450 80B1 isencoded by the CYP80B1 gene. In examples, cytochrome P450 80B1 catalyzesthe reaction of N-methylcoclaurine→3′-hydroxy-N-methylcoclaurine, asreferenced in FIG. 5. An engineered host cell may be modified to includeconstitutive expression of the cytochrome P450 80B1 gene in theengineered host cell. Additionally or alternatively, the engineered hostcell may be modified to synthetically regulate the expression of thecytochrome P450 80B1 gene in the engineered host cell. In examples, theengineered host cell may be modified to incorporate a copy, copies, oradditional copies, of the cytochrome P450 80B1 gene. Additionally oralternatively, the engineered host cell may be modified to incorporatethe introduction of a strong promoter element for the overexpression ofthe cytochrome P450 80B1 gene within the engineered host cell. In somecases, the CYP80B1 gene may be codon optimized for expression inSaccharomyces cerevisiae. The cytochrome P450 80B1 gene may be derivedfrom P. somniferum, E. californica, T. flavum, or another species. Insome examples, the P450 80B1 gene may be 77% similar to the naturallyoccurring gene.

[FOL2] In some examples, the engineered host cell may modify theexpression of the enzyme GTP cyclohydrolase. GTP cyclohydrolase isencoded by the FOL2 gene. In some examples, GTP cyclohydrolase catalyzesthe reaction of GTP→dihydroneopterin triphosphate, as referenced inFIG. 1. The engineered host cell may be modified to include constitutiveoverexpression of the FOL2 gene in the engineered host cell. Theengineered host cell may also be modified to include native regulation.Additionally or alternatively, the engineered host cell may be modifiedto synthetically regulate the expression of the FOL2 gene in theengineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of theFOL2 gene. Additionally or alternatively, the engineered host cell maybe modified to incorporate the introduction of a strong promoter elementfor the overexpression of the FOL2 gene within the engineered host cell.The FOL2 gene may be derived from Saccharomyces cerevisiae, Homosapiens, Mus musculus, or another species. In some examples, the FOL2gene may be 100% similar to the naturally occurring gene.

[PTPS] In some examples, the engineered host cell may modify theexpression of the enzyme 6-pyruvoyl tetrahydrobiopterin (PTP) synthase.Pyruvoyl tetrahydrobiopterin synthase is encoded by the PTPS gene. Insome examples, 6-pyruvoyl tetrahydrobiopterin synthase catalyzes thereaction of dihydroneopterin triphosphate→PTP, as referenced in FIG. 1.The engineered host cell may be modified to include constitutiveexpression of the PTPS gene in the engineered host cell. Additionally oralternatively, the engineered host cell may be modified to syntheticallyregulate the expression of the PTPS gene in the engineered host cell. Inexamples, the engineered host cell may be modified to incorporate acopy, copies, or additional copies, of the PTPS gene. Additionally oralternatively, the engineered host cell may be modified to incorporatethe introduction of a strong promoter element for the overexpression ofthe PTPS gene within the engineered host cell. In some cases, the PTPSgene may be codon optimized for expression in Saccharomyces cerevisiae.The PTPS gene may be derived from Rattus norvegicus, Homo sapiens, Musmusculus, or another species. In some examples, the PTPS gene may be 80%similar to the naturally occurring gene.

[SepR] In some examples, the engineered host cell may modify theexpression of the enzyme sepiapterin reductase. Sepiapterin reductase isencoded by the SepR gene. In some examples, sepiapterin reductasecatalyzes the reaction of PTP→BH₄, as referenced in FIG. 1. Theengineered host cell may be modified to include constitutive expressionof the SepR gene in the engineered host cell. Additionally oralternatively, the engineered host cell may be modified to syntheticallyregulate the expression of the SepR gene in the engineered host cell. Inexamples, the engineered host cell may be modified to incorporate acopy, copies, or additional copies, of the SepR gene. Additionally oralternatively, the engineered host cell may be modified to incorporatethe introduction of a strong promoter element for the overexpression ofthe SepR gene within the engineered host cell. In some cases, the SepRgene may be codon optimized for expression in Saccharomyces cerevisiae.The SepR gene may be derived from Rattus norvegicus, Homo sapiens, Musmusculus, or another species. In some examples, the SepR gene may be 72%similar to the naturally occurring gene.

[PCD] In some examples, the engineered host cell may modify theexpression of the enzyme 4a-hydroxytetrahydrobiopterin(pterin-4α-carbinolamine) dehydratase. 4a-hydroxytetrahydrobiopterindehydratase is encoded by the PCD gene. In some examples,4a-hydroxytetrahydrobiopterin dehydratase catalyzes the reaction of4a-hydroxytetrahydrobiopterin→H₂O+quinonoid dihydropteridine, asreferenced in FIG. 1. The engineered host cell may be modified toinclude constitutive expression of the PCD gene in the engineered hostcell. Additionally or alternatively, the engineered host cell may bemodified to synthetically regulate the expression of the PCD gene in theengineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of the PCDgene. Additionally or alternatively, the engineered host cell may bemodified to incorporate the introduction of a strong promoter elementfor the overexpression of the PCD gene within the engineered host cell.In some cases, the PCD gene may be codon optimized for expression inSaccharomyces cerevisiae. The PCD gene may be derived from Rattusnorvegicus, Homo sapiens, Mus musculus, or another species. In someexamples, the PCD gene may be 79% similar to the naturally occurringgene.

[QDHPR] In some examples, the engineered host cell may modify theexpression of the enzyme quinonoid dihydropteridine reductase. Quinonoiddihydropteridine reductase is encoded by the QDHPR gene. In someexamples, quinonoid dihydropteridine reductase catalyzes the reaction ofquinonoid dihydropteridine→BH₄, as referenced in FIG. 1. The engineeredhost cell may be modified to include constitutive expression of theQDHPR gene in the engineered host cell. Additionally or alternatively,the engineered host cell may be modified to synthetically regulate theexpression of the QDHPR gene in the engineered host cell. In examples,the engineered host cell may be modified to incorporate a copy, copies,or additional copies, of the QDHPR gene. Additionally or alternatively,the engineered host cell may be modified to incorporate the introductionof a strong promoter element for the overexpression of the QDHPR genewithin the engineered host cell. In some cases, the QDHPR gene may becodon optimized for expression in Saccharomyces cerevisiae. The QDHPRgene may be derived from Rattus norvegicus, Homo sapiens, Mus musculus,or another species. In some examples, the QDHPR gene may be 75% similarto the naturally occurring gene.

[DHFR] In some examples, the engineered host cell may modify theexpression of the enzyme dihydrofolate reductase. Dihydrofolatereductase is encoded by the DHFR gene. In some examples, dihydrofolatereductase catalyzes the reaction of 7,8-dihydrobiopterin(BH₂)→5,6,7,8-tetrahydrobiopterin (BH₄), as referenced in FIG. 1. Thisreaction may be useful in recovering BH₄ as a co-substrate for theconversion of tyrosine to L-DOPA, as illustrated in FIG. 5. Theengineered host cell may be modified to include constitutive expressionof the DHFR gene in the engineered host cell. Additionally oralternatively, the engineered host cell may be modified to syntheticallyregulate the expression of the DHFR gene in the engineered host cell. Inexamples, the engineered host cell may be modified to incorporate acopy, copies, or additional copies, of the DHFR gene. Additionally oralternatively, the engineered host cell may be modified to incorporatethe introduction of a strong promoter element for the overexpression ofthe DHFR gene within the engineered host cell. In some cases, the DHFRgene may be codon optimized for expression in Saccharomyces cerevisiae.The DHFR gene may be derived from Rattus norvegicus, Homo sapiens, oranother species. In some examples, the DHFR gene may be 77% similar tothe naturally occurring gene.

[CYP-COR] As discussed above with regard to epimerizing 1-BIAs, theengineered host cell may modify the expression of a BIA epimerase. TheBIA epimerase is encoded by the CYP-COR gene (e.g., CYP82Y2-COR gene).The CYP-COR gene may also be referred to as the DRS-DRR gene. In someexamples, the BIA epimerase catalyzes the conversion of(S)-1-BIA→(R)-1-BIA, as referenced in FIG. 7. In particular, FIG. 7illustrates a biosynthetic scheme for conversion of L-tyrosine tomorphinan alkaloids, in accordance with embodiments of the invention.FIG. 7 provides the use of the enzymes CPR, cytochrome P450 reductase;CYP-COR, cytochrome P450 CYP82Y1-like codeinone reductase-like fusion;SalSyn, salutaridine synthase; SalR, salutaridine reductase; SalAT,salutaridinol 7-O-acetyltransferase; T6ODM, thebaine 6-O-demethylase;COR, codeinone reductase; and CODM, codeine-O-demethylase.

The engineered host cell may be modified to include constitutiveexpression of the CYP-COR gene in the engineered host cell. Additionallyor alternatively, the engineered host cell may be modified tosynthetically regulate the expression of the CYP-COR gene in theengineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of theCYP-COR gene. Additionally or alternatively, the engineered host cellmay be modified to incorporate the introduction of a strong promoterelement for the overexpression of the CYP-COR gene within the engineeredhost cell. The CYP-COR gene may be derived from Papaver bracteatum,Papaver somniferum, Papaver setigerum, Chelidonium majus, or anotherspecies. In some examples, the CYP-COR gene may be 77% similar to thenaturally occurring gene.

[CPR] In some examples, the engineered host cell may modify theexpression of the enzyme cytochrome P450 reductase. The cytochrome P450reductase is encoded by the CPR gene. In some examples, the cytochromeP450 reductase catalyzes the reaction of (R)-reticuline→salutaridine, asreferenced in FIG. 7. Additionally, the cytochrome P450 reductasecatalyzes other reactions such as those described in FIGs. throughoutthe application. The engineered host cell may be modified to includeconstitutive expression of the CPR gene in the engineered host cell.Additionally or alternatively, the engineered host cell may be modifiedto synthetically regulate the expression of the CPR gene in theengineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of the CPRgene. Additionally or alternatively, the engineered host cell may bemodified to incorporate the introduction of a strong promoter elementfor the overexpression of the CPR gene within the engineered host cell.The CPR gene may be derived from E. californica, P. somniferum, H.sapiens, S. cerevisiae, A. thaliana, or another species. In someexamples, the CPR gene may be 100% similar to the naturally occurringgene.

[SalSyn] In some examples, the engineered host cell may modify theexpression of the enzyme salutaridine synthase. The salutaridinesynthase is encoded by the SalSyn gene. In some examples, thesalutaridine synthase catalyzes the reaction of(R)-reticuline→salutaridine, as referenced in FIG. 7. The engineeredhost cell may be modified to include constitutive expression of theSalSyn gene in the engineered host cell. Additionally or alternatively,the engineered host cell may be modified to synthetically regulate theexpression of the SalSyn gene in the engineered host cell. In examples,the engineered host cell may be modified to incorporate a copy, copies,or additional copies, of the SalSyn gene. Additionally or alternatively,the engineered host cell may be modified to incorporate the introductionof a strong promoter element for the overexpression of the SalSyn genewithin the engineered host cell. In some cases, the SalSyn gene may becodon optimized for expression in Saccharomyces cerevisiae. In someexamples the SalSyn may be modified at the N-terminus. The SalSyn genemay be derived from Papaver somniferum, Papaver spp, Chelidonium majus,or another species. In some examples, the SalSyn gene may be 78% similarto the naturally occurring gene.

[SalR] In some examples, the engineered host cell may modify theexpression of the enzyme salutaridine reductase. Salutaridine reductaseis encoded by the SalR gene. In some examples, salutaridine reductasereversibly catalyzes the reaction of salutaridinol→salutaridine, asreferenced in FIG. 7. The engineered host cell may be modified toinclude constitutive expression of the SalR gene in the engineered hostcell. Additionally or alternatively, the engineered host cell may bemodified to synthetically regulate the expression of the SalR gene inthe engineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of theSalR gene. Additionally or alternatively, the engineered host cell maybe modified to incorporate the introduction of a strong promoter elementfor the overexpression of the SalR gene within the engineered host cell.In some cases, the SalR gene may be codon optimized for expression inSaccharomyces cerevisiae. The SalR gene may be derived from Papaversomniferum, Papaver bracteatum, Papaver spp., Chelidonium majus, oranother species. In some examples, the SalR gene may be 80-100% similarto the naturally occurring gene.

[SalAT] In some examples, the engineered host cell may modify theexpression of the enzyme acetyl-CoA:salutaridinol 7-O-acetyltransferase.Acetyl-CoA:salutaridinol 7-O-acetyltransferase is encoded by the SalATgene. In some examples, acetyl-CoA:salutaridinol 7-O-acetyltransferasecatalyzes the reaction ofacetyl-CoA+salutaridinol→CoA+7-O-acetylsalutaridinol, as referenced inFIG. 7. The engineered host cell may be modified to include constitutiveexpression of the SalAT gene in the engineered host cell. Additionallyor alternatively, the engineered host cell may be modified tosynthetically regulate the expression of the SalAT gene in theengineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of theSalAT gene. Additionally or alternatively, the engineered host cell maybe modified to incorporate the introduction of a strong promoter elementfor the overexpression of the SalAT gene within the engineered hostcell. In some cases, the SalAT gene may be codon optimized forexpression in Saccharomyces cerevisiae. The SalAT gene may be derivedfrom Papaver somniferum, Papaver bracteatum, Papaver orientate, Papaverspp., or another species. In some examples, the SalAT gene may be 77-80%similar to the naturally occurring gene.

[T6ODM] In some examples, the engineered host cell may modify theexpression of the enzyme thebaine 6-O-demethylase. Thebaine 6-Odemethylase is encoded by the T6ODM gene. In some examples, thebaine6-O-demethylase catalyzes the reaction of thebaine→neopinone, asreferenced in FIG. 7. Once the neopinone has been produced, theneopinone may be converted to codeinone. The conversion of neopinone 4codeinone may occur spontaneously. Alternatively, the conversion ofneopinone 4 codeinone may occur as a result of a catalyzed reaction. Inother examples, the T6ODM enzyme may catalyze the O-demethylation ofsubstrates other than thebaine. For example, T6ODM may O-demethylateoripavine to produce morphinone. Alternatively, T6ODM may catalyze theO-demethylation of BIAs within the 1-benzylisoquinoline, protoberberine,or protopine classes such as papaverine, canadine, and allocryptopine,respectively. The engineered host cell may be modified to includeconstitutive expression of the T6ODM gene in the engineered host cell.Additionally or alternatively, the engineered host cell may be modifiedto synthetically regulate the expression of the T6ODM gene in theengineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of theT6ODM gene. Additionally or alternatively, the engineered host cell maybe modified to incorporate the introduction of a strong promoter elementfor the overexpression of the T6ODM gene within the engineered hostcell. In some cases, the T6ODM gene may be codon optimized forexpression in Saccharomyces cerevisiae. The T6ODM gene may be derivedfrom Papaver somniferum, or another species. In some examples, the T6ODMgene may be 76.2% similar to the naturally occurring gene.

[COR] In some examples, the engineered host cell may modify theexpression of the enzyme codeinone reductase. Codeinone reductase isencoded by the COR gene. In some examples, codeinone reductase catalyzesthe reaction of codeinone to codeine, as referenced in FIG. 7. In somecases, codeinone reductase can catalyze the reaction of neopinone toneopine. In other examples, COR can catalyze the reduction of othermorphinans including hydrocodone→dihydrocodeine,14-hydroxycodeinone→14-hydroxycodeine, andhydromorphone→dihydromorphine. The engineered host cell may be modifiedto include constitutive expression of the COR gene in the engineeredhost cell. Additionally or alternatively, the engineered host cell maybe modified to synthetically regulate the expression of the COR gene inthe engineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of the CORgene. Additionally or alternatively, the engineered host cell may bemodified to incorporate the introduction of a strong promoter elementfor the overexpression of the COR gene within the engineered host cell.In some cases, the COR gene may be codon optimized for expression inSaccharomyces cerevisiae. Additionally or alternatively, the COR genemay be modified with the addition of targeting sequences formitochondria, vacuole, endoplasmic reticulum, or a combination thereof.The COR gene may be derived from Papaver somniferum, or another species.In some examples, the COR gene may be 76-78% similar to the naturallyoccurring gene. In examples, the COR gene may be 76.8%, 77.0%, 77.3%, or77.7% similar to the naturally occurring gene.

[CODM] In some examples, the engineered host cell may modify theexpression of the enzyme codeine O-demethylase. Codeine O-demethylase isencoded by the CODM gene. In some examples, codeine O-demethylasecatalyzes the reaction of codeine to morphine, as referenced in FIG. 7.Codeine O-demethylase can also catalyze the reaction of neopine toneomorphine. Codeine O-demethylase can also catalyze the reaction ofthebaine to oripavine. In other examples, CODM may catalyze theO-demethylation of BIAs within the 1-benzylisoquinoline, aporphine, andprotoberberine classes such as reticuline, isocorydine, and scoulerine,respectively. In other examples, the CODM enzyme may catalyze anO,O-demethylenation reaction to cleave the methylenedioxy bridgestructures in protopines. The engineered host cell may be modified toinclude constitutive expression of the CODM gene in the engineered hostcell. Additionally or alternatively, the engineered host cell may bemodified to synthetically regulate the expression of the CODM gene inthe engineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of theCODM gene. Additionally or alternatively, the engineered host cell maybe modified to incorporate the introduction of a strong promoter elementfor the overexpression of the CODM gene within the engineered host cell.In some cases, the CODM gene may be codon optimized for expression inSaccharomyces cerevisiae. Additionally or alternatively, the CODM genemay be modified with the addition of targeting sequences formitochondria. The CODM gene may be derived from Papaver somniferum,Papaver spp., or another species. In some examples, the CODM gene may be75% similar to the naturally occurring gene. In examples, the CODM genemay be 75.2% similar to the naturally occurring gene.

[BBE] In some examples, the engineered host cell may modify theexpression of the enzyme berberine bridge enzyme. The berberine bridgeenzyme is encoded by the BBE gene. In some examples, berberine bridgeenzyme catalyzes the reaction of (S)-reticuline→(S)-scoulerine. Theengineered host cell may be modified to include constitutive expressionof the BBE gene in the engineered host cell. Additionally oralternatively, the engineered host cell may be modified to syntheticallyregulate the expression of the BBE gene in the engineered host cell. Inexamples, the engineered host cell may be modified to incorporate acopy, copies, or additional copies, of the BBE gene. Additionally oralternatively, the engineered host cell may be modified to incorporatethe introduction of a strong promoter element for the overexpression ofthe BBE gene within the engineered host cell. The BBE gene may bederived from Papaver somniferum, Argemone mexicana, Eschscholziacalifornica, Berberis stolonifera, Thalictrum flavum subsp. glaucum,Coptis japonica, Papaver spp., or another species. In some examples, theBBE gene may be 99% similar to the naturally occurring gene.

[S9OMT] In some examples, the engineered host cell may modify theexpression of the enzyme S-adenosyl-L-methionine:(S)-scoulerine9-O-methyltransferase. S-adenosyl-L-methionine:(S)-scoulerine9-O-methyltransferase is encoded by the S9OMT gene. In some examples,S-adenosyl-L-methionine:(S)-scoulerine 9-O-methyltransferase catalyzesthe reaction ofS-adenosyl-L-methionine+(S)-scoulerine→S-adenosyl-L-homocysteine+(S)-tetrahydrocolumbamine.The engineered host cell may be modified to include constitutiveexpression of the S9OMT gene in the engineered host cell. Additionallyor alternatively, the engineered host cell may be modified tosynthetically regulate the expression of the S9OMT gene in theengineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of theS9OMT gene. Additionally or alternatively, the engineered host cell maybe modified to incorporate the introduction of a strong promoter elementfor the overexpression of the S9OMT gene within the engineered hostcell. In some cases, the S9OMT gene may be codon optimized forexpression in Saccharomyces cerevisiae. The S9OMT gene may be derivedfrom Thalictrum flavum subsp. glaucum, Coptis japonica, Coptischinensis, Papaver somniferum, Thalictrum spp., Coptis spp., Papaverspp., or another species. In some examples, the S9OMT gene may be 100%similar to the naturally occurring gene. In examples, the S9OMT gene maybe 80% similar to the naturally occurring gene.

[CAS] In some examples, the engineered host cell may modify theexpression of the enzyme (S)-canadine synthase. (S)-canadine synthase isencoded by the CAS gene. In some examples, (S)-canadine synthasecatalyzes the reaction of (S)-tetrahydrocolumbamine 4 (S)-canadine. Theengineered host cell may be modified to express the CAS gene in theengineered host cell. The engineered host cell may be modified toinclude constitutive expression of the CAS gene in the engineered hostcell. Additionally or alternatively, the engineered host cell may bemodified to synthetically regulate the expression of the CAS gene in theengineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of the CASgene. Additionally or alternatively, the engineered host cell may bemodified to incorporate the introduction of a strong promoter elementfor the overexpression of the CAS gene within the engineered host cell.The CAS gene may be derived from Thalictrum flavum subsp. glaucum,Coptis japonica, Thalictrum spp., Coptis spp., or another species. Insome examples, the CAS gene may be 100%

[STOX] In some examples, the engineered host cell may modify theexpression of the enzyme (S)-tetrahydroprotoberberine oxidase.(S)-tetrahydroprotoberberine oxidase is encoded by the STOX gene. Insome examples, (S)-tetrahydroprotoberberine oxidase catalyzes thereaction of (S)-tetrahydroberberine+2 O₂→berberine+2 H₂O₂. Theengineered host cell may be modified to include constitutive expressionof the STOX gene in the engineered host cell. Additionally oralternatively, the engineered host cell may be modified to syntheticallyregulate the expression of the STOX gene in the engineered host cell. Inexamples, the engineered host cell may be modified to incorporate acopy, copies, or additional copies, of the STOX gene. Additionally oralternatively, the engineered host cell may be modified to incorporatethe introduction of a strong promoter element for the overexpression ofthe STOX gene within the engineered host cell. In some examples the STOXmay be modified at the N-terminus. In some cases, the STOX gene may becodon optimized for expression in Saccharomyces cerevisiae. The STOXgene may be derived from Berberis wilsonae, Coptis japonica, Berberisspp., Coptis spp., or another species. In some examples, the STOX genemay be 78% similar to the naturally occurring gene.

[TNMT] In some examples, the engineered host cell may modify theexpression of the enzyme tetrahydroprotoberberine-N-methyltransferase.Tetrahydroprotoberberine-N-methyltransferase is encoded by the TNMTgene. In some examples, tetrahydroprotoberberine-N-methyltransferasecatalyzes the reaction of canadine→N-methylcanadine. In some examples,tetrahydroprotoberberine-N-methyltransferase catalyzes the reaction ofnoroxymorphone→naloxone, as referenced in FIG. 26.

In other examples, tetrahydroprotoberberine-N-methyltransferasecatalyzes the reaction of stylopine→cis-N-methylstylopine. Theengineered host cell may be modified to include constitutive expressionof the TNMT gene in the engineered host cell. Additionally oralternatively, the engineered host cell may be modified to syntheticallyregulate the expression of the TNMT gene in the engineered host cell. Inexamples, the engineered host cell may be modified to incorporate acopy, copies, or additional copies, of the TNMT gene. Additionally oralternatively, the engineered host cell may be modified to incorporatethe introduction of a strong promoter element for the overexpression ofthe TNMT gene within the engineered host cell. In some cases, the TNMTgene may be codon optimized for expression in Saccharomyces cerevisiae.The TNMT gene may be derived from Papaver somniferum, Eschscholziacalifornica, Papaver bracteatum, Argemone mexicana, or another species.In some examples, the TNMT gene may be 100% similar to the naturallyoccurring gene. In examples, the TNMT gene may be 81% similar to thenaturally occurring gene.

[CFS] In some examples, the engineered host cell may modify theexpression of the enzyme cheilanthifoline synthase. Cheilanthifolinesynthase is encoded by the CFS gene. In examples, cheilanthifolinesynthase catalyzes the reaction of scoulerine→cheilanthifoline. Anengineered host cell may be modified to include constitutive expressionof the CFS gene in the engineered host cell. Additionally oralternatively, the engineered host cell may be modified to syntheticallyregulate the expression of the CFS gene in the engineered host cell. Inexamples, the engineered host cell may be modified to incorporate acopy, copies, or additional copies, of the CFS gene. Additionally oralternatively, the engineered host cell may be modified to incorporatethe introduction of a strong promotor element for the overexpression ofthe CFS gene within the engineered host cell. The CFS gene may bederived from P. somniferum, E. californica, A. mexicana, or anotherspecies. In some examples, the CFS gene may be 77%, 78%, or 79% similarto the naturally occurring gene. Additionally, the CFS gene may be codonoptimized for expression in Saccharomyces cerevisiae.

[STS] In some examples, the engineered host cell may modify theexpression of the enzyme stylopine synthase. Stylopine synthase isencoded by the STS gene. In examples, stylopine synthase catalyzes thereaction of cheilanthifoline→stylopine. An engineered host cell may bemodified to include constitutive expression of the STS gene in theengineered host cell. Additionally or alternatively, the engineered hostcell may be modified to synthetically regulate the expression of the STSgene in the engineered host cell. In examples, the engineered host cellmay be modified to incorporate a copy, copies, or additional copies, ofthe STS gene. Additionally or alternatively, the engineered host cellmay be modified to incorporate the introduction of a strong promotorelement for the overexpression of the STS gene within the engineeredhost cell. The STS gene may be derived from P. somniferum, E.californica, A. mexicana, or another species. In some examples, the STSgene may be 76%, 78%, or 79% similar to the naturally occurring gene.Additionally, the STS gene may be codon optimized for expression inSaccharomyces cerevisiae.

[MSH] In some examples, the engineered host cell may modify theexpression of the enzyme cis-N-methylstylopine 14-hydroxylase.Cis-N-methylstylopine 14-hydroxylase is encoded by the MSH gene. Inexamples, cis-N-methylstylopine 14-hydroxylase catalyzes the reaction ofcis-N-methylstylopine→protopine. An engineered host cell may be modifiedto include constitutive expression of the MSH gene in the engineeredhost cell. Additionally or alternatively, the engineered host cell maybe modified to synthetically regulate the expression of the MSH gene inthe engineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of the MSHgene. Additionally or alternatively, the engineered host cell may bemodified to incorporate the introduction of a strong promotor elementfor the overexpression of the MSH gene within the engineered host cell.The MSH gene may be derived from P. somniferum or another species. Insome examples, the MSH gene may be 79% similar to the naturallyoccurring gene. Additionally, the MSH gene may be codon optimized forexpression in Saccharomyces cerevisiae.

[P6H] In some examples, the engineered host cell may modify theexpression of the enzyme protopine-6-hydroxylase.Protopine-6-hydroxylase is encoded by the P6H gene. In examples,protopine-6-hydroxylase catalyzes the reaction ofProtopine→6-hydroxyprotopine. An engineered host cell may be modified toinclude constitutive expression of the P6H gene in the engineered hostcell. Additionally or alternatively, the engineered host cell may bemodified to synthetically regulate the expression of the P6H gene in theengineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of the P6Hgene. Additionally or alternatively, the engineered host cell may bemodified to incorporate the introduction of a strong promotor elementfor the overexpression of the CFS gene within the engineered host cell.The P6H gene may be derived from P. somniferum, E. californica, oranother species. In some examples, the P6H gene may be 79% similar tothe naturally occurring gene. Additionally, the P6H gene may be codonoptimized for expression in Saccharomyces cerevisiae.

[DBOX] In some examples, the engineered host cell may modify theexpression of the enzyme dihydrobenzophenanthridine oxidase.Dihydrobenzophenanthridine oxidase is encoded by the DBOX gene. Inexamples, dihydrobenzophenanthridine oxidase catalyzes the reaction ofdihydrosanguinarine→sanguinarine. An engineered host cell may bemodified to include constitutive expression of the DBOX gene in theengineered host cell. Additionally or alternatively, the engineered hostcell may be modified to synthetically regulate the expression of theDBOX gene in the engineered host cell. In examples, the engineered hostcell may be modified to incorporate a copy, copies, or additionalcopies, of the DBOX gene. Additionally or alternatively, the engineeredhost cell may be modified to incorporate the introduction of a strongpromotor element for the overexpression of the DBOX gene within theengineered host cell. The DBOX gene may be derived from P. somniferum oranother species. In some examples, the DBOX gene may be 100% similar tothe naturally occurring gene. Additionally, the DBOX gene may be codonoptimized for expression in Saccharomyces cerevisiae.

[morA] In some examples, the engineered host cell may modify theexpression of the enzyme morphine dehydrogenase. Morphine dehydrogenaseis encoded by the morA gene. In some examples, morphine dehydrogenasecatalyzes the reaction of morphine→morphinone, as referenced in FIG. 8.In other examples, morphine dehydrogenase catalyzes the reaction ofcodeinone→codeine, also as referenced in FIG. 8. FIG. 8 illustrates abiosynthetic scheme for production of semi-synthetic opiods, inaccordance with embodiments of the invention. In particular, FIG. 8illustrates extended transformations of thebaine in yeast byincorporating morA, morphine dehydrogenase; and morB, morphinereductase. FIG. 30 illustrates an additional transformation of thebaine,in accordance with embodiments of the invention.

The engineered host cell may be modified to include constitutiveexpression of the morA gene in the engineered host cell. Additionally oralternatively, the engineered host cell may be modified to syntheticallyregulate the expression of the morA gene in the engineered host cell. Inexamples, the engineered host cell may be modified to incorporate acopy, copies, or additional copies, of the morA gene. Additionally oralternatively, the engineered host cell may be modified to incorporatethe introduction of a strong promoter element for the overexpression ofthe morA gene within the engineered host cell. In some cases, the morAgene may be codon optimized for expression in Saccharomyces cerevisiae.The morA gene may be derived from Pseudomonas putida or another species.In some examples, the morA gene may be 73.7% similar to the naturallyoccurring gene.

[morB] In some examples, the engineered host cell may modify theexpression of the enzyme morphinone reductase. Morphinone reductase isencoded by the morB gene. In some examples, morphinone reductasecatalyzes the reaction of codeinone→hydrocodone, as referenced in FIG.8. In other examples, morphinone reductase catalyzes the reaction ofmorphinone→hydromorphone, also as referenced in FIG. 8. In otherexamples, morphinone reductase catalyzes the reaction14-hydroxycodeinone→oxycodone. The engineered host cell may be modifiedto include constitutive expression of the morB gene in the engineeredhost cell. Additionally or alternatively, the engineered host cell maybe modified to synthetically regulate the expression of the morB gene inthe engineered host cell. In examples, the engineered host cell may bemodified to incorporate a copy, copies, or additional copies, of themorB gene. Additionally or alternatively, the engineered host cell maybe modified to incorporate the introduction of a strong promoter elementfor the overexpression of the morB gene within the engineered host cell.In some cases, the morB gene may be codon optimized for expression inSaccharomyces cerevisiae. The morB gene may be derived from Pseudomonasputida or another species. In some examples, the morB gene may be 67.2%similar to the naturally occurring gene.

[CYP80A1] In some examples, the engineered host cell may express theenzyme berbamunine synthase. Berbamunine synthase is encoded by the genefor cytochrome P450 enzyme 80A1 (CYP80A1). In some examples, CYP80A1catalyzes the reaction(S)—N-methylcoclaurine+(R)—N-methylcoclaurine→berbamunine. In otherexamples, CYP80A1 catalyzes the reaction(R)—N-methylcoclaurine+(R)—N-methylcoclaurine→guattegaumerine. In otherexamples, CYP80A1 catalyzes the reaction(R)—N-methylcoclaurine+(S)-coclaurine→2′norberbamunine. The engineeredhost cell may be modified to include constitutive expression of theCYP80A1 gene in the engineered host cell. Additionally or alternatively,the engineered host cell may be modified to synthetically regulate theexpression of the CYP80A1 gene in the engineered host cell. In examples,the engineered host cell may be modified to incorporate a copy, copies,or additional copies, of the CYP80A1 gene. Additionally oralternatively, the engineered host cell may be modified to incorporatethe introduction of a strong promoter element for the overexpression ofthe CYP80A1 gene within the engineered host cell. In some cases, theCYP80A1 gene may be codon optimized for expression in Saccharomycescerevisiae. The CYP80A1 gene may be derived from Berberis stolonifera oranother species. In some examples, the CYP80A1 gene may be 76% similarto the naturally occurring gene.

[PODA] In some example, the engineered host cell may express the enzymeprotopine O-dealkylase. Protopine O-dealkylase is encoded by the genePODA. In some examples, PODA catalyzes the O,O-demethylenation ofprotoberberines and protopines such as canadine, stylopine, berberine,cryptopine, allocryptopine, and protopine. In some examples, PODAcatalyzes the O-demethylation of BIAs including tetrahydropapaverine,tetrahydropalmatine, and cryptopine. The engineered host cell may bemodified to include constitutive expression of the PODA gene in theengineered host cell. Additionally or alternatively, the engineered hostcell may be modified to synthetically regulate the expression of thePODA gene in the engineered host cell. In examples, the engineered hostcell may be modified to incorporate a copy, copies, or additionalcopies, of the PODA gene. Additionally or alternatively, the engineeredhost cell may be modified to incorporate the introduction of a strongpromoter element for the overexpression of the PODA gene within theengineered host cell. In some cases, the PODA gene may be codonoptimized for expression in Saccharomyces cerevisiae. The PODA gene maybe derived from Papaver somniferum or other species. In some examples,the PODA gene may be 70-100% similar to the naturally occurring gene.

[BM3] In some examples, the engineered host cell may express the enzymeBM3. BM3 is a Bacillus megaterium cytochrome P450 involved in fatty acidmonooxygenation in its native host. In some cases BM3 N-demethylates anopioid to produce a nor-opioid, as referenced in FIG. 27. It is alsoreadily expressed as an active heterologous enzyme in yeast andbacteria. BM3 has several advantages as a biosynthetic enzyme includingthat it is soluble, comes with a fused reductase partner protein, andcan readily be engineered to accept new substrates. Additionally, Table6 illustrates variants of BM3 N-demethylase.

Examples of the aforementioned genes can be expressed from a number ofdifferent platforms in the host cell, including plasmid (2μ, ARS/CEN),YAC, or genome. In addition, examples of the aforementioned genesequences can either be native or codon optimized for expression in thedesired heterologous host (e.g., Saccharomyces cerevisiae).

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the invention inany fashion. The present examples, along with the methods describedherein are presently representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1: Tyrosine Hydroxylase Mutants Improve Reticuline Production inEngineered Yeast Strains

Tyrosine hydroxylase from R. norvegicus was yeast codon optimized,synthesized, and cloned into a low-copy plasmid. Single mutants (W166Y,E332D, S40D and R37ER38E), double mutants (W166Y and E332D, W166Y andS40D, W166Y and R37ER38E), and one triple mutant (W166Y, R37ER38E, andE332D) were generated through site-directed mutagenesis. Each TyrHmutant was expressed from a low-copy plasmid with the GPD promoter in ayeast strain containing the following mutations to central metabolism(as described in U.S. Provisional Patent Application Ser. No.61/899,496): ARO4^(FBR), ΔZWF1, and GPD-TKL1 promoter replacement. Inaddition, the strain expressed a chromosomally integrated copy of DOPAdecarboxylase (DODC) from P. putida, four chromosomally integrated genesfrom R. norvegicus that generate the cosubstrate tetrahydrobiopterin(pyruvoyl tetrahydropterin synthase, PTPS; sepiapterin reductase, SepR;pterin 4a-carbinolamine dehydratase, PCD; dihydropteridine reductase,QDHPR), norcoclaurine synthase (NCS) from C. japonica expressed from alow-copy plasmid with a GPD promoter, and five genes for thebiosynthesis of reticuline from norcoclaurine (P. somniferum6-O-methyltransferase, Ps6OMT; P. somniferum coclaurineN-methyltransferase, PsCNMT; E. californica cytochrome P450 80B1,EcCYP80B1; P. somniferum cytochrome P450 NADPH reductase, PsCPR; and P.somniferum 3′hydroxy-N-methylcoclaurine 4′-O-methyltransferase,Ps4′OMT). The strains harboring TyrH mutants were grown in selectivedefined media (YNB) lacking tyrosine with 2% dextrose for 96 hours, andthe production of reticuline was measured in the media via LC-MS/MS inMRM mode with the transition 330 m/z to 137 m/z. FIG. 9 shows theresults of this assay and demonstrates that TyrH mutants can improvereticuline production by as much as 5-fold when compared to wild-typeTyrH. As such, FIG. 9 illustrates tyrosine hydroxylase mutants thatimprove reticuline production from sugar in engineered yeast strains, inaccordance with embodiments of the invention.

Example 2: Expression of DHFR Improves Tyrosine Hydroxylase Activity inEngineered Yeast Strains

Dihydrofolate reductase (DHFR) from R. norvegicus was yeast codonoptimized, synthesized, and cloned into a low-copy plasmid under thecontrol of a GPD promoter. DHFR was coexpressed with wild-type RnTyrH(low-copy plasmid with a GPD promoter) in a yeast strain containing thefollowing mutations to central metabolism (as described in U.S.Provisional Patent Application Ser. No. 61/899,496): ARO4^(FBR), ΔZWF1,and GPD-TKL1 promoter replacement. In addition, the strain expressedfour chromosomally integrated genes from R. norvegicus that generate thecosubstrate tetrahydrobiopterin (pyruvoyl tetrahydropterin synthase,PTPS; sepiapterin reductase, SepR; pterin 4a-carbinolamine dehydratase,PCD; dihydropteridine reductase, QDHPR). The strains expressing DHFR andwild-type RnTyrH were grown in selective defined media (YNB) lackingtyrosine with 2% dextrose for 96 hours, and the production of L-DOPA wasmeasured in the media via LC-MS/MS in MRM mode with the transition 198m/z to 152 m/z. Expression of DHFR with wild-type RnTyrH increasesL-DOPA production by 1.8-fold, as illustrated in FIG. 10. As such, FIG.10 illustrates coexpression of dihydrofolate reductase (DHFR) thatimproves L-DOPA production by tyrosine hydroxylase in engineered yeaststrains, in accordance with embodiments of the invention.

Example 3: Addition of Antioxidants to Growth Media Improve TyrosineHydroxylase Activity in Engineered Yeast Strains

A yeast strain containing the following mutations to central metabolism(as described in U.S. Provisional Patent Application Ser. No.61/899,496): ARO4^(FBR), ΔZWF1, and GPD-TKL1 promoter replacement andexpressing four chromosomally integrated genes from R. norvegicus thatgenerate the cosubstrate tetrahydrobiopterin (pyruvoyl tetrahydropterinsynthase, PTPS; sepiapterin reductase, SepR; pterin 4a-carbinolaminedehydratase, PCD; dihydropteridine reductase, QDHPR) as well aswild-type RnTyrH from a low-copy plasmid under the control of the GPDpromoter was grown in selective defined media (YNB) lacking tyrosinewith 2% galactose and 2 mM ascorbic acid for 96 hours.

The production of L-DOPA was measured in the media via LC-MS/MS in MRMmode with the transition 198 m/z to 152 m/z. The addition of 2 mMascorbic acid improves L-DOPA production with wild-type RnTyrH by1.8-fold. In addition, the concentration BH₄ intermediates were measuredwith LC-MS/MS in MRM mode with the following transitions: B, 238 m/z to178 m/z; BH2, 240 m/z to 165 m/z and BH4, 242 m/z to 166 m/z. Theaddition of ascorbic acid also increases BH₄ in the media, whichindicates the oxidation of BH₄ to BH₂ is prevented.

Accordingly, FIG. 11A illustrates addition of antioxidants to culturemedia that improves L-DOPA production by tyrosine hydroxylase inengineered yeast strains and (B) addition of antioxidants to culturemedia that increase BH₄ levels, in accordance with embodiments of theinvention. In particular, FIG. 11A illustrate a wild-type RnTyrH(expressed from a low-copy plasmid under the control of a GPD promoter)was expressed in a yeast strain containing the following mutations tocentral metabolism (as described in U.S. Provisional Patent ApplicationSer. No. 61/899,496): ARO4^(FBR), ΔZWF1, and GPD-TKL1 promoterreplacement. In addition, the strain expressed four chromosomallyintegrated genes from R. norvegicus that generate the cosubstratetetrahydrobiopterin (pyruvoyl tetrahydropterin synthase, PTPS;sepiapterin reductase, SepR; pterin 4a-carbinolamine dehydratase, PCD;dihydropteridine reductase, QDHPR). The strains expressing wild-typeRnTyrH was grown in selective defined media (YNB) lacking tyrosine with2% dextrose, with and without 2 mM ascorbic acid (aa) for 96 hours. Theproduction of L-DOPA was measured in the media via LC-MS/MS in MRM modewith the transition 198 m/z to 152 m/z. Additionally, FIG. 11Billustrates, in the same strain described in FIG. 11A, the concentrationof the BH₄ intermediate was measured in the media of strains grown withand without 2 mM ascorbic acid (aa) with LC-MS/MS in MRM mode with thefollowing transition: BH₄, 242 m/z to 166 m/z.

Example 4. Identification of an Epimerase Enzyme

To identify an epimerase enzyme suitable for performing theepimerization reactions of the methods disclosed herein, a cytochromeP450 oxidase 82Y1-like domain and a codeinone reductase-like domain wereidentified in a single open reading frame (CYP-COR) in publicallyavailable plant transcriptomes. The CYP-COR fusions were identified froma BLAST search of the 1000 Plants Project (Matasci, et al. 2014.Gigascience. 3: 17) and PhytoMetaSyn (Facchini, et al. 2012. TrendsBiotechnol. 30: 127-31; Xiao, et al. 2013. J. Biotechnol. 166: 122-34)transcriptomes using blastn with the query being the sequence of apreviously published COR-silencing VIGS construct that resulted inreticuline accumulation (Wijekoon and Facchini. 2012. Plant J. 69:1052-63). Once one CYP-COR fusion sequence was observed as a hit, thatsequence was translated and the amino acid sequence was used as thequery for a second search of both databases with tblastn. A phylogenetictree of the CYP-COR fusion enzymes identified from the databases isprovided in FIG. 13. The sequences were identified from The 1000 PlantsProject and PhytoMetaSyn transcriptome databases based on abioinformatic search. Additionally, an example amino acid sequence isprovided in FIG. 4, as discussed above. Additionally, Table 1 listsvarious examples of amino acid sequences identified for this CYP-CORenzyme, which come from various plants including Papaver somniferum(opium poppy), Papaver setigerum (poppy of Troy), Papaver bracteatum(Iranian poppy), and Chelidonium majus (greater celandine).

Example 5. Epimerization of (S)-Reticuline to (R)-Reticuline in anEngineered Non Plant Host Cell

Non-plant host cells were engineered to heterologously express enzymesdescribed herein. For instance, yeast strains (Saccharomyces cerevisiae)were engineered to heterologously express the identified epimerasesdescribed in Example 4 and to verify their function in the context ofthis microbial host. The yeast-codon optimized DNA coding sequences forthe partial amino acid sequences pbr.PBRST1PF_4328 andpbr.PBRST1PF_89405 were synthesized in-frame with the yeast-codonoptimized coding sequence for amino acids 1-40 of SSDU-2015634 (Table 1)to generate CYP-COR_4328 and CYP-COR_89405, respectively. These CYP-CORcoding sequences were cloned into a low-copy plasmid harboring a URA3selection marker and expressed from the TDH3 promoter. The plasmids weretransformed into yeast strains that harbored an expression cassette fora cytochrome P450 reductase (P_(TEF1)-ATR1 or P_(TEF1)-PsCPRv2)integrated into the chromosome. These yeast strains harboring the twoplasmids were grown in synthetic complete media with the appropriateddrop out solution (-Ura-Trp). The yeast strains were fed (S)-reticulineand BIA metabolites were analyzed after 72 hours of growth by LC-MS/MSanalysis.

Example 6. Production of Salutaridine from (S)-Reticuline in anEngineered Yeast Cell

Yeast strains (Saccharomyces cerevisiae) were engineered toheterologously express the identified epimerases described in Example 4and to verify their function in the context of this microbial host. Theyeast-codon optimized DNA coding sequences for the partial amino acidsequences pbr.PBRST1PF_4328 and pbr.PBRST1PF_89405 were synthesizedin-frame with the yeast-codon optimized coding sequence for amino acids1-40 of SSDU-2015634 (Table 1) to generate CYP-COR_4328 andCYP-COR_89405, respectively. These CYP-COR coding sequences were clonedinto a low-copy plasmid harboring a URA3 selection marker and expressedfrom the TDH3 promoter. The salutaridine synthase (SalSyn) codingsequence was cloned into a low-copy plasmid harboring a TRP1 selectionmarker and expressed from the TDH3 promoter. The plasmids weretransformed into yeast strains that harbored an expression cassette fora cytochrome P450 reductase (P_(TEF1)-ATR1 or P_(TEF1)-PsCPRv2)integrated into the chromosome. These yeast strains harboring the twoplasmids were grown in synthetic complete media with the appropriateddrop out solution (-Ura-Trp). The yeast strains were fed (S)-reticulineand BIA metabolites were analyzed after 72 hours of growth by LC-MS/MSanalysis. The analysis indicated that the engineered yeast cells wereable to convert (S)-reticuline to (R)-reticuline, which was then actedon by salutaridine synthase to form salutaridine, a 4-ring promorphinanalkaloid (FIG. 7, FIG. 14). Salutaridine synthase has been previouslyshown to act on (R)-reticuline and have no observable activity on(S)-reticuline (Gesell, et al. 2009. J. Biol. Chem. 284: 24432-42).

As shown in FIG. 7, CYP-COR catalyzes the conversion of (S)-reticulineto (R)-reticuline, which is then acted on by salutaridine synthase tomake the promorphinan alkaloid salutaridine. FIG. 14 illustrates (A)chromatogram traces showing reticuline and salutaridine for twoepimerase variants (CYP-COR_89405, CYP-COR_4328) and a standard. FIG. 14also illustrates (B) the same chromatogram traces for salutaridine in(A) as replotted to demonstrate co-elution with the standard. In thisexperiment, the yeast contains two low-copy CEN/ARS plasmids with URA3and TRP1 selective markers, TDH3 promoters, and the CYP-COR and SalSyncoding sequences. Yeast were grown from freshly transformed colonies in3 mL selective media overnight, back-diluted into 3.5 mL media to OD0.8, grown 7 hours, pelleted, and then resuspended into pH 7.4 HEPESbuffer with 100 μM (S)-reticuline (Specs). After 16 hours on a spinnerat 30° C., the yeast were pelleted and the buffer supernatant wasanalyzed by LC-MS/MS. Each trace is from a single sample representativeof 2. Peaks are normalized such that the largest peak in allchromatograms is 100%.

Example 7. Production of (R)-Reticuline from Racemic Norlaudanosoline inan Engineered Non-Plant Host Cell

Yeast strains (Saccharomyces cerevisiae) were engineered toheterologously express the identified epimerases described in Example 4and to verify their function in the context of this microbial host. Theyeast-codon optimized DNA coding sequence CYP-COR_89405 described inExample 5 was cloned into a low-copy plasmid harboring a URA3 selectionmarker and expressed from the TDH3 promoter. This plasmid wastransformed into yeast strains that harbored expression cassettes for acytochrome P450 reductase (P_(TEF1)-ATR1 or P_(TEF1)-PsCPRv2) and threemethyltransferases (Papaver somniferumnorcoclaurine-6-O-methyltransferase, coclaurine N-methyltransferase, and3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase, all expressed fromP_(TEF1)) integrated into the chromosome. This yeast strain harboringthe plasmid was grown in synthetic complete media with the appropriateddrop out solution (-Ura). The yeast strain was fed racemicnorlaudanosoline and BIA metabolites were analyzed after 72 hours ofgrowth by LC-MS/MS analysis. For chiral characterization, reticuline wasconcentrated from yeast media by pelleting 5 mL yeast culture and adding120 mg XAD-4 resin to 4 mL supernatant, incubating on rotator overnightat room temperature, and eluting with 0.5 mL methanol. The concentratewas fractionated by reverse-phase HPLC (Pursuit XRs-C18, 5 μm, 50 mm×10mm) with isocratic 15% methanol with 0.1% formic acid over 6.5 min witha flow rate of 5 mL/min and injection volume of 40-50 μL. Peak-basedfractions were collected at approximately 4.5 min. Fractions werepooled, freeze-dried, and resuspended in 0.5 mL isopropanol. Dependingon concentration, 0.5-5 μL were injected onto a chiral column(Phenomenex Lux cellulose-1, 3 μm, 150 mm×2 mm) and separated withisocratic 72% N-hexane, 28% isopropanol, 0.1% diethylamine with a flowrate of 0.3 mL/min and detection by MS and 250 nm UV. MS detection wasperformed with an Agilent 6320 Ion Trap mass spectrometer with ESIsource gas temperature 350° C., gas flow of 10 L/min, nebulizer pressure40 PSI and isolation of m/z 330.1 with width 1.0. The retention time ofreticuline peaks was compared to that of authentic (S)-reticuline and(R)-reticuline standards. The analysis indicated that the engineeredyeast cells containing the CYP-COR plasmid were able to convert racemicnorlaudanosoline to (R)-reticuline, while engineered yeast cells with anempty plasmid produced exclusively (S)-reticuline (FIG. 15).

Example 8: Protein Engineering of Salutaridine Synthase to Improve itsProcessing and Activity when Expressed in a Microbial Host

Heterologous proteins may be incorrectly processed when expressed in arecombinant host, for example, plant proteins such as cytochrome P450enzymes expressed in microbial production hosts. For example,salutaridine synthase, which converts (R)-reticuline to salutaridine,undergoes N-linked glycosylation when heterologously expressed in yeast(FIG. 16A and FIG. 16B). The observed N-linked glycosylation pattern onsalutaridine synthase is not observed when the enzyme is expressed inplants and is indicative of incorrect N-terminal sorting of the nascentSalSyn transcript, which reduces the activity of the enzyme in theheterologous microbial host. Thus, protein engineering directed atcorrecting N-terminal sorting of the nascent transcript and therebyremoving the N-linked glycosylation pattern will result in improvedactivity of the salutaridine synthase enzyme in the recombinantproduction host.

For example, N-terminal alpha-helices from cheilanthifoline synthase(CFS) were used to replace N-terminal alpha-helices from salutaridinesynthase (SalSyn, FIG. 17). Junction points for these fusions wereselected based on secondary structure motifs of CFS and SalSyn or basedon amino acid alignments of CFS and SalSyn. The fusions were cloned byamplifying the N-terminal fragment from CFS and C-terminal fragment fromSalSyn with 15-40 nucleotides of overlap with the other fragment, andthen assembled with each other and a vector backbone by Gibson assemblyto form the complete fusion open reading frame (Gibson, et al. 2009. NatMethods. 6: 343-5).

As another example, the coding sequence for the cytochrome P450 domainfrom salutaridine synthase was placed directly into the P450 encodingregion of other stably expressed cytochrome P450s such as the BM3enzyme. For example, the conserved cytochrome P450 domain of thesalutaridine synthase and the cytochrome P450 domain from an engineeredvariant of the Bacillus megaterium P450 monooxygenase CYP102A1 (BM3,(Michener and Smolke. 2012. Metab. Eng. 14: 306-16)) were identified byNCBI conserved domain search. Primers were designed to fuse the codingsequence of the first few amino acids of the BM3 to the coding sequencefor the P450 domain of the salutaridine synthase, followed by the codingsequence for BM3 domains C-terminal to the P450 domain. As before, thisconstruct was assembled via Gibson assembly.

The engineered salutaridine synthase protein fusions were analyzed byWestern Blot analysis to confirm full-length expression and modificationto or elimination of N-linked glycosylation patterns in yeast (FIG. 16Aand FIG. 16B). The salutaridine synthase enzyme and protein fusions wereC-terminally tagged with the human influenza hemagglutinin (HA) epitopeand cloned into expression plasmids appropriate for yeast and plantexpression. For yeast, the enzyme coding sequences were cloned into alow-copy yeast/E. coli shuttle vector harboring a URA3 selection markerand expressed from the TDH3 promoter. For plants, the sequences werecloned into an E. coli/Agrobacterium tumefaciens shuttle vector withkanamycin resistance and the Cauliflower mosaic virus (CaMV) 35Spromoter with flanking 5′ and 3′-untranslated regions from Cowpea mosaicvirus RNA-2 for transient plant expression via Agrobacteriumtumefaciens-infiltration. Yeast engineered to express salutaridinesynthase exhibited a banding pattern indicative of N-linkedglycosylation. We confirmed that this pattern was due to N-linkedglycosylation by performing site-directed mutagenesis on theglycosylation site. In contrast, plant expression of this enzyme did notresult in a banding pattern indicative of N-linked glycosylation, asseen in FIG. 16A. Although the N-linked glycosylation sites wereunmodified, the engineered salutaridine synthase protein fusions werenot N-glycosylated when expressed in yeast, as seen in FIG. 16B. ByWestern blot, we demonstrated that the yeast-expressed fusion enzymeswere present as a single band, similar to the expression observed forthe plant-expressed parent enzyme, indicating that the mis-processing ofthe nascent protein in yeast that resulted in N-linked glycosylation wasrepaired by the engineered fusions.

The engineered salutaridine synthase protein fusions were analyzed forimproved enzyme activity when heterologously expressed in yeast. Codingsequences for salutaridine synthase and the engineered fusions werecloned into a low-copy plasmid harboring a URA3 selection marker andexpressed from the TDH3 promoter. The yeast have P_(TEF1)-PsCPRv2integrated into the TRP1 locus and contain a single low-copy plasmidwith the URA3 selective marker and the salutaridine synthase codingsequence with the TDH3 promoter. Yeast were grown from freshlytransformed colonies in 1 mL selective media (-Ura) overnight andback-diluted 1:20 into 0.5 mL selective media in 96-well plates with 10∞M (R)-reticuline (Toronto Research Chemicals). After 72-96 hours in theshaking incubator, the yeast were pelleted and the media supernatant wasanalyzed by LC-MS/MS. The analysis indicated that the engineeredsalutaridine synthase enzymes exhibited improved activity relative tothat of the wild-type sequence when heterologously expressed in yeast(FIG. 18).

FIG. 18 illustrates salutaridine synthase codon-optimization andengineered fusions that improve activity in yeast, in accordance withembodiments of the invention. As seen in FIG. 18, a black bar indicatesa native wild-type sequence for salutaridine synthase, PsCYP719B1. Greybars with black borders are yeast codon-optimized variants from Papaversomniferum and a newly identified sequence from Papaver bracteatum. Thediagonally patterned bar indicates the most improved engineered fusion,which is based on the P. bracteatum sequence. Error bars indicate therange of at least two biological replicates. Natural, syntheticcodon-optimized, and/or protein engineered variants of salutaridinesynthase from P. bracteatum, P. somniferum, or P. setigerum (or relatedplant) may be used in these engineered strains.

The engineered salutaridine synthase protein fusions can be used in thecontext of a biosynthetic pathway to increase production of downstreambenzylisoquinoline alkaloid products. In one example, yeast wereengineered to heterologously express yeast codon optimized genesencoding an engineered salutaridine synthase fusion, P. bracteatumsalutaridine reductase, and P. somniferum salutaridinol7-O-acetyltransferase. The three expression cassettes(P_(TDH3)-D94yPsSS, P_(TPI1)-yPbSalR, P_(TEF1)-yPsSalAT) were assembledinto a yeast artificial chromosome (YAC) with a TRP1 section marker. TheYAC was placed into yeast that harbored an expression cassette for acytochrome P450 reductase (P_(TEF1)-ATR1 or P_(TEF1)-yPsCPRv2)integrated into the chromosome. The yeast strains were grown insynthetic complete media with the appropriated drop out solution (-Trp)and fed (R)-reticuline. BIA metabolites were analyzed after 96 hours ofgrowth through LC-MS/MS analysis. The analysis indicates that yeaststrains engineered with the engineered salutaridine synthase enzymes andother pathway enzymes produce the morphinan alkaloid thebaine, asillustrated in (A) of FIG. 19.

Accordingly, FIG. 19 illustrates (A) an LC/MS-MS analysis of small scalebatch fermentation in which engineered yeast catalyze the conversion of(R)-reticuline to thebaine, in accordance with embodiments of theinvention. As provided in (A) of FIG. 19, yeast strains are engineeredto have a P_(TEF1)-ATR1 expression cassette integrated into the TRP1locus and contain a single yeast artificial chromosome with the TRP1selective marker and three expression cassettes:P_(TDH3)-yEcCFS¹⁻⁸³-yPsSS⁹⁵⁻⁵⁰⁵, P_(TPI1)-yPbSalR, andP_(TEF1)-yPsSalAT. Yeast were grown from freshly transformed colonies in3 mL selective media overnight and back-diluted 1:20 into 0.5 mLselective media (-Trp) in culture tubes with 100 μM (R)-reticuline(Toronto Research Chemicals). After 72 hours in the shaking incubator,the yeast were pelleted and the media supernatant was analyzed byLC-MS/MS. Chromatogram traces show thebaine produced by this strain andsalutaridinol and salutaridine accumulated, along with standards. Thesetraces are representative of two samples.

Example 9: Protein Engineering of Enzymes in the Downstream MorphinanBranch to Improve Production of Morphinan Products from a HeterologousMicrobial Host

In one embodiment of the invention, pathway enzymes are engineered toexhibit increased activity to increase production of the BIA ofinterest. In this example, mutations were introduced into the openreading frame of a particular pathway enzyme by amplification withMutazyme II (see Table 11). Sufficient template DNA was included in theamplification reaction to result in a mutation rate of 1-4 nucleotidesubstitutions per gene. The mutagenized library was cloned into thepYES1L vector by gap repair directly in yeast. In several instances,yeast strains selected for library expression contained integratedcopies of genes that generate the substrate of the mutagenized enzyme.For example, a library of CODM variants was transformed into a strainwith integrated copies of T6ODM and COR1.3 and fed thebaine in theculture medium. Expression of T6ODM and COR1.3 in these strains ensuredthat codeine and neopine would be available as substrates for eachintroduced CODM variant. Individual colonies were inoculated into96-well plates and cultured 96 hours then assayed for production oftheir product by liquid chromatograph mass spectrometry (LC-MS). In theexample of the CODM library, the products screened for were morphine andneomorphine. In each screen, variants with enhanced BIA production weresequenced and re-cloned for validation. Table 11 includes a summary ofmutated enzyme variants identified through the screens that resulted inincreased BIA production in yeast.

FIG. 20 shows data of the validated enhanced activity of one of thesemutants. In particular, FIG. 20 illustrates generation of a CODM enzymevariant exhibiting enhanced activity in yeast through random mutagenesisand screening, in accordance with embodiments of the invention. Alibrary of CODM variants was generated by mutagenizing the coding regionby error-prone PCR. A variant identified by screening of this library,CODM^(N35S,G335V), was re-cloned and expressed in a yeast strainharboring integrated copies of T6ODM and COR1.3. This strain and anothercontrol strain expressing wild-type CODM were cultured in liquid mediumwith 1 mM thebaine. After 96 hours the culture medium was analyzed forCODM activity by LC-MS. Variant CODM^(N35S,G335V) produced 1.4× moremorphine and 2.6× more neomorphine than a strain expressing wild-typeCODM.

Example 10: Optimization of Expression and Growth Conditions to ImproveBenzylisoquinoline Alkaloid Production from a Heterologous MicrobialHost

Benzylisoquinoline alkaloid production from an engineered microbial hostcan be further improved by optimizing the expression of pathway enzymesand growth conditions. In one example, the expression of salutaridinol7-O-acetyltransferase was altered in yeast by expressing the enzyme froma series of different promoters. The yeast were engineered toheterologously express yeast codon-optimized genes encoding P.somniferum salutaridinol 7-O-acetyltransferase from different promoters(as provided in FIG. 21A). Two expression cassettes (P_(TPI1)-yPbSalR,P_(X)-yPsSalAT) were assembled into a yeast artificial chromosome (YAC)with a TRP1 section marker. The YAC was placed into yeast and cells weregrown in synthetic complete media with the appropriated drop outsolution (-Trp) and fed salutaridine. BIA metabolites were analyzedafter 72 hours of growth by LC-MS/MS analysis. The optimization ofpathway enzyme expression level can result in increased production ofthe morphinan alkaloid thebaine (as provided in FIG. 21A).

Optimization of strain cultivation conditions, including but not limitedto sugar source, growth temperature, and pH, can be used to increaseproduction of benzylisoquinoline alkaloids from engineered yeast strains(as provided in FIG. 21B and FIG. 21C). In one example, pH was varied toincrease thebaine production from engineered yeast strains. Twoexpression cassettes (P_(TPI1)-yPbSalR, P_(TEF1)-yPsSalAT) wereassembled into a yeast artificial chromosome (YAC) with a TRP1 sectionmarker. The YAC was placed into yeast and cells were grown in syntheticcomplete media with the appropriated drop out solution (-Trp),resuspended in buffer at pH 5.7-9, and fed salutaridine. BIA metaboliteswere analyzed after 16 hours of incubation by LC-MS/MS analysis. Levelsof the 4-ring promorphinan alkaloid salutaridinol and the 5-ringmorphinan alkaloid thebaine increased as a function of increasing pH (asprovided in (B) of FIG. 21).

In another example, temperature, sugar, and media buffer content werevaried to increase thebaine production from engineered yeast strains.Three expression cassettes (P_(TDH3)-D94yPsSS, P_(TPI1)-yPbSalR,P_(TEF1)-yPsSalAT) were assembled into a yeast artificial chromosome(YAC) with a TRP1 section marker. The YAC was placed into yeast thatharbored an expression cassette for a cytochrome P450 reductase(P_(TEF1)-ATR1 or P_(TEF1)-yPsCPRv2) integrated into the chromosome. Theyeast strains were grown in synthetic complete media with theappropriated drop out solution (-Trp) and fed (R)-reticuline. BIAmetabolites were analyzed after 72 hours of growth by LC-MS/MS analysis.The analysis indicates the microbial production of the morphinanalkaloid thebaine increases under certain cultivation conditions(buffered media with dextrose at 30° C., as provided in FIG. 21C).

Accordingly, FIG. 21A, FIG. 21B, and FIG. 21C illustrate fermentationoptimization for conversion of (R)-reticuline to thebaine by engineeredyeast, in accordance with embodiments of the invention. LC/MS-MSanalysis of whole cell buffered assay of (A) SalAT promoter variants,(B) SalR and SalAT strain grown under different pH conditions, and (C)optimization of sugar source, growth temperature, and media buffercontent. (A) Yeast strains engineered to contain a single yeastartificial chromosome with the TRP1 selective marker and two expressioncassettes: P_(TPI1)-yPbSalR and P_(X)-yPsSalAT with varied SalATpromoters. Yeast were grown from freshly transformed colonies in 3 mLselective media overnight and back-diluted 1:20 into 0.5 mL media inculture tubes with 100 μM salutaridine (Specs). After 72 hours in theshaking incubator, the yeast were pelleted and the media supernatant wasanalyzed by LC-MS/MS. (B) Yeast strains engineered to contain a singleyeast artificial chromosome with the TRP1 selective marker and twoexpression cassettes: P_(TPI1)-yPbSalR and P_(TEF1)-yPsSalAT. Yeast weregrown from freshly transformed colonies in 3 mL selective mediaovernight, back-diluted into 3.5 mL media to OD 0.8, grown 7 hours,pelleted, and then resuspended into pH 5.7 MOPS, or pH 7, 8, or 9 Trisbuffer with 10 μM salutaridine (Specs). After 16 hours on a spinner at30° C., the yeast were pelleted and the buffer supernatant was analyzedby LC-MS/MS. Error bars represent the range of two samples. (C)Optimization of sugar source, growth temperature, and media buffercontent. In this experiment, the yeast strains are engineered to haveP_(TEF1)-ATR1 integrated into the TRP1 locus and contain a single yeastartificial chromosome with the TRP1 selective marker and threeexpression cassettes: P_(TDH3)-yEcCFS¹⁻⁸³-yPsSS⁹⁵⁻⁵⁰⁵, P_(TPI1)-yPbSalR,and P_(TEF1)-yPsSalAT. Yeast were grown from freshly transformedcolonies in 3 mL selective media overnight and back-diluted 1:20 into0.5 mL media in culture tubes with 100 μM (R)-reticuline (TorontoResearch Chemicals). After 72 hours in the shaking incubator, the yeastwere pelleted and the media supernatant was analyzed by LC-MS/MS.

Example 11: Yeast Engineered for the Production of Thebaine from anEarly 1-Benzylisoquinoline Alkaloid Scaffold

Yeast strains can be engineered for the production of the morphinanalkaloid thebaine, or morphinan alkaloids derived from thebaine, fromearly 1-benzylisoquinoline alkaloids. As an example, the engineeredyeast strains can produce the morphinan alkaloid products from racemicor (S)-norcoclaurine or racemic or (S)-norlaudanosoline (FIGS. 5, 6, and7, and (B) of 23). Yeast strains are engineered to produce(S)-reticuline from (S)-norcoclaurine or racemic or (S)-norlaudanosolineby the integration of three or five expression cassettes into the yeastgenome. To produce (S)-reticuline from racemic or (S)-norlaudanosoline,the integrated expression cassettes encode Papaver somniferumnorcoclaurine 6-O-methyltransferase (Ps6OMT, EC 2.1.1.128),4′-O-methyltransferase (Ps4′OMT, EC 2.1.1.116), andcoclaurine-N-methyltransferase (CNMT, EC 2.1.1.140), each with a TEF1promoter (Hawkins and Smolke. 2008. Nat. Chem. Biol. 4: 564-73). Toproduce (S)-reticuline from racemic or (S)-norcoclaurine, the strainfurther harbors integrated expression cassettes for yeastcodon-optimized Eschscholzia californica N-methylcoclaurine3′-hydroxylase (yEcCYP80B1, EC 1.14.13.71) and ATR1 or yPsCPRv2cytochrome P450 reductase expressed from the TDH3 or TEF1 promoter (CPR,EC 1.6.2.4). These strains are further engineered to incorporateepimerization-catalyzing enzymes (e.g., CYP-COR), salutaridine synthase,salutaridine reductase, and salutaridinol acetyltransferase to convertracemic or (S)-norcoclaurine or racemic or (S)-norlaudanosoline to themorphinan alkaloid thebaine, or morphinan alkaloids derived fromthebaine (FIG. 7). As an alternative to expression of anepimerization-catalyzing enzyme, 6OMT, 4′OMT, CNMT, and/or CYP80B1 maybe engineered such that rac-reticuline is produced fromrac-norcoclaurine or rac-norlaudanosoline.

In one example, a yeast strain was engineered to convertrac-norlaudanosoline to thebaine. The yeast strain harbors integratedexpression cassettes encoding Ps6OMT, Ps4′OMT, CNMT, and yPsCPRv2, eachwith a TEF1 promoter. Four expression cassettes(P_(TDH3)-yEcCFS¹⁻⁸³-yPsSS⁹⁵⁻⁵⁰⁵, P_(TPI1)-yPbSalR, P_(TEF1)-yPsSalAT,P_(HXT7)-CYP-COR_89405) were assembled into a yeast artificialchromosome (YAC) with a TRP1 selective marker in this strain. The yeaststrain harboring the YAC and integrated cassettes was grown in syntheticcomplete media with the appropriated drop out solution (-Trp) and 1 mMrac-norlaudanosoline substrate. After 96 hours of growth, the media wasanalyzed for BIA metabolites by LC-MS/MS analysis. Nearly 200 nMthebaine was detected ((B) of FIG. 19). Other engineered salutaridinesynthase variants may also be used in this strain (FIG. 18, Example 8).

Example 12: Platform Yeast Strains Engineered for the Production ofReticuline from L-Tyrosine

A platform yeast strain that produces the key branch point BIAintermediate (S)-reticuline from L-tyrosine was constructed (FIG. 5).Specifically, four multi-gene expression constructs were integrated intothe genome of a yeast strain. The composition of the four constructs isindicated in FIG. 22. Each construct is comprised of 4 or 5 genesexpressed from strong constitutive promoters. Genes are positioned ateach locus as complete expression cassettes comprising a promoter, geneopen reading frame, and terminator as specified in the annotations abovethe schematic. The schematic shows the orientation of each expressioncassette by the direction of the arrow representing a given gene.Selectable markers are italicized in the annotation and represented bygrey arrows in the schematic. Each selection marker is flanked by loxPsites to allow removal of the marker from the locus. Additionally, eachconstruct has a selectable marker flanked by loxP sites so that it canbe removed by Cre recombinase.

In the first integration construct, four heterologous genes from Rattusnorvegicus are integrated into the YBR197C locus together with a G418selection marker (KanMX). RnPTPS, RnSepR, RnPCD, and RnQDHPR arerequired to synthesize and regenerate tetrahydrobiopterin (BH₄) from theyeast endogenous folate synthesis pathway. Each gene is codon optimizedfor expression in yeast.

In the second integration construct, four heterologous genes areintegrated into the HIS3 locus together with the HISS selection marker.Rattus norvegicus tyrosine hydroxylase (RnTyrH) converts tyrosine toL-DOPA using the cosubstrate BH₄ generated by the preceding integrationconstruct. The RnTyrH gene can be any of the wild-type or improvedmutants which confer enhanced activity (e.g., W166Y, R37E, and R38E,Example 1). A second Rattus norvegicus gene, RnDHFR, encodes an enzymethat reduces dihydrobiopterin (an oxidation product of BH₄) to BH₄, inthis way increasing the availability of this cosubstrate. Also includedin the third construct is PpDODC from Pseudomonas putida, an enzyme thatconverts L-DOPA to dopamine. The fourth enzyme is CjNCS from Coptisjaponica, which condenses 4-HPA and dopamine to make norcoclaurine. Eachgene is codon optimized for expression in yeast.

In the third integration construct, five heterologous genes from plantsand the LEU2 selection marker are integrated into the locus YDR514C.Ps6OMT, Ps4′OMT, and PsCNMT are methyltransferases from Papaversomniferum and are expressed as native plant nucleotide sequences. Afourth P. somniferum gene, yPsCPRv2, is codon optimized for yeast andencodes a reductase that supports the activity of a cytochrome P450 fromEschscholzia californica, EcCYP80A1. EcCYP80A1 is expressed as itsnative plant nucleotide sequence. The enzymes encoded in this constructperform two O-methylations, an N-methylation, and a hydroxylation toproduce reticuline from the norcoclaurine produced by the precedingintegration construct.

In the final integration construct, additional copies of Saccharomycescerevisiae endogenous genes ARO4^(Q166K), ARO7^(T226I), TKL1, and ARO10are integrated into the ARO4 locus together with a hygromycin resistanceselection marker. ARO4^(Q166K) and ARO7^(T226I) are feedback-resistantmutants of ARO4 and ARO10 which each encode a single base pairsubstitution relative to the wild-type sequence. TKL1 and ARO10 areidentical to the native yeast genes, but are expressed behind strongpromoters. Aro4p and Aro7p are enzymes in the biosynthesis of aromaticamino acids including tyrosine. Removing feedback inhibition from theseenzymes results in upregulation of endogenous tyrosine biosynthesis.Overexpression of Tkl1p upregulates the pentose phosphate pathwayresulting in enhanced supply of erythrose 4-phosphate (E4P), a precursorfor tyrosine. Overexpression of Aro10p increases the production of4-HPA.

Platform yeast strains can be constructed with any number of the fourexpression cassettes. Specifically, platform yeast strains wereconstructed with integration constructs 1-4 and integration constructs1-3. In the latter strain in which the tyrosine over-productionconstruct (construct 4) is excluded, additional tyrosine may be suppliedin the culture medium to support the biosynthesis of reticuline.Additional genetic modifications may be incorporated into the platformstrains to support production of downstream BIAs and increased flux toBIA biosynthesis.

The yeast strains were grown in synthetic complete media with theappropriated amino acid drop out solution at 25 and 30° C. BIAmetabolites in the media supernatant were analyzed after 48 and 96 hoursof growth by LC-MS/MS analysis.

Example 13: Yeast Engineered for the Production of Thebaine and OtherMorphinan Alkaloids from L-Tyrosine

Yeast strains can be engineered for the production of the morphinanalkaloid thebaine, or morphinan alkaloids derived from thebaine, fromearly precursors such as tyrosine. As an example, the platform yeaststrains described in Example 12 can be further engineered to produce themorphinan alkaloid products from L-tyrosine (FIG. 7).

The platform yeast strain producing (S)-reticuline from L-tyrosine (seedescription in Example 12) was further engineered to incorporateepimerization-catalyzing enzymes, such as the newly identified CYP-COR,salutaridine synthase, salutaridine reductase, and salutaridinolacetyltransferase to convert the biosynthesized (S)-reticuline to themorphinan alkaloid thebaine, or morphinan alkaloids derived fromthebaine (FIG. 7). Three expression cassettes(P_(TDH3)-yEcCFS¹⁻²⁶-yPbSS³³⁻⁵⁰⁴, P_(TPI1)-yPbSalR, P_(TEF1)-yPsSalAT)were assembled into a yeast artificial chromosome (YAC) with a TRP1selective marker directly in the platform yeast strain. Other engineeredsalutaridine synthase variants may also be incorporated into the YAC(FIG. 18, Example 8). The resulting yeast strain was also transformedwith a low-copy CEN/ARS plasmid with a URA3 selective marker, TDH3promoter, and a CYP-COR coding sequence.

The yeast strains harboring the YAC, low-copy plasmid, and integratedcassettes were grown in synthetic complete media with the appropriateddrop out solution (-Ura-Trp) at 25 and 30° C. After 96 hours of growth,the media was analyzed for BIA metabolites by LC-MS/MS analysis. Furtherculture optimization with respect to temperature, carbon source, pHcondition, and media composition was performed to improve BIAproduction.

Additional genetic modifications can be introduced into the yeaststrains to produce morphinan alkaloids derived from thebaine (FIG. 7).In one example, the expression cassettes P_(ADH1)-T6ODM-T_(ADH1),P_(HXT7)-COR-T_(PGK1), and P_(TEF1)-CODM-T_(CYC1) were directlyassembled and integrated into the trp1 locus of the thebaine-producingyeast strain (Thodey et al., 2014). In another example, these yeaststrains can be further engineered to produce additional morphinealkaloids by directly assembling the expression cassettesP_(GPD)-morA-T_(CYC1), P_(PGK1)-morB-T_(PHO5) and integrating thisconstruct into the ura3 locus on the chromosome (Thodey et al., 2014).

Example 14: O-Demethylation of Opioid Molecules

For high throughput screening of demethylation reactions a purpald assaywas used. For example, demethylation catalyzed by 2-oxoglutaratedependent dioxygenases produces formaldehyde as a product as shown inthe generalized chemical equation: [substrate]+2-oxoglutarate+O2[product]+formaldehyde+succinate+CO2. Purpald reagent in alkalineconditions undergoes a color change in the presence of formaldehyde thatcan be quantified to concentrations as low as 1 nM with aspectrophotometer at 510 nm.

An important step in the production of nor-opioid compounds is theO-demethylation of molecules such as oxycodone (see FIG. 23). Toidentify enzymes capable of performing this step, sequences from Table 3were subjected to codon optimization for expression in S. cerevisiae,and ordered as synthetic genes (from Integrated DNA Technologies). Codonoptimized sequences were cloned into expression vectors pA24, pA25, orpA26 (or similar vectors), shown in FIG. 28, which harbor promotersequences of varying strength, by gap repair in the Cen.PK2 yeast hoststrain, according to standard molecular biology procedures. Individualcolonies were isolated and verified by PCR and sequencing (ELIMbiopharmaceuticals).

FIG. 28 illustrates plasmid/YAC vectors for enzyme expression andengineering, in accordance with embodiments of the invention. Candidateand engineered enzymes were cloned into these vectors for expression inS. cerevisiae strains. Examples of pA24, pA25, and pA26 sequences areprovided in Table 7.

Strains expressing putative O-demethylase enzymes were then tested forbasal levels of activity on the various substrates listed in FIG. 23. Todetect activity, cell cultures were grown in selective medium (asdescribed above), lysed by glass bead disruption, and incubated withsubstrates in the presence of redox molecules and other cofactors (suchas NADH, NADPH, and iron, at different concentrations depending on theenzyme requirements). O-demethylation of substrates, including but notlimited to those listed in FIG. 23, was then detected by analysis viaLC-MS of experimental and control samples (such as quantified amounts ofoxycodone and oxymorphone, for example).

To identify engineered enzymes with improved O-demethylation activity,sequences encoding enzymes listed in Table 3 were subjected to randommutagenesis and then screened via a high-throughput colorimetric assay.Initial libraries were generated by error-prone PCR using Mutazyme II(Agilent Technologies), and variants were cloned into the pA24 (orsimilar) vector by gap repair in the Cen.PK2 screening host. Strainsexpressing mutated enzymes, with either individual mutations orcombinations of mutations introduced by gene shuffling or other methods,were grown in selective medium in 96-well plate format under varyingfermentation conditions (different media components, pH, andtemperature, for example), pelleted, and lysed by glass bead disruption.Lysates were incubated with substrates (listed in FIG. 23) and assayedfor formaldehyde production in the purpald assay. Enzymes with improvedO-demethylation activity were verified by directly measuringO-demethylated product (oxymorphone, for example) formation in theculture medium by LC-MS.

Example 15: N-Demethylation of Opioid Molecules

N-demethylase activity removes the N-methyl group present in opioidsubstrate molecules (such as oxymorphone) and produces a nor-opioidcompound (such as noroxymorphone), an important intermediate in theultimate biosynthesis of nal-opioids. To identify enzymes capable ofperforming this step, sequences from Table 4 were subjected to codonoptimization for expression in S. cerevisiae, and ordered as syntheticgenes (from Integrated DNA Technologies). Codon optimized sequences werecloned into expression vectors pA24, pA25, or pA26 (or similar vectors),shown in FIG. 28, which harbor promoter sequences of varying strength,by gap repair in the Cen.PK2 yeast host strain, according to standardmolecular biology procedures. Individual colonies were isolated andverified by PCR and sequencing (ELIM biopharmaceuticals).

Strains expressing putative O-demethylase enzymes were then tested forbasal levels of activity on the various substrates listed in FIG. 24. Todetect activity, cell cultures were grown in selective medium (asdescribed above), lysed by glass bead disruption, and incubated withsubstrates in the presence of redox molecules and other cofactors (NADH,NADPH, and iron, for example). N-demethylation of substrates was thendetected by analysis via LC-MS of experimental and control samples.

To identify engineered enzymes with improved N-demethylation activity,sequences encoding enzymes listed in Table 4 were subjected to randommutagenesis and then screened via a high-throughput colorimetric assay.Initial libraries were generated by error-prone PCR using Mutazyme II(Agilent Technologies), and variants were cloned into the pA24 (orsimilar) vector by gap repair in the Cen.PK2 screening host. Strainsexpressing mutated enzymes, with either individual mutations orcombinations of mutations introduced by gene shuffling or other methods,were grown in selective medium in 96-well plate format under varyingfermentation conditions (different media components, pH, andtemperature, for example), pelleted, and lysed by glass bead disruption.Lysates were incubated with substrates (listed in FIG. 24) and assayedfor formaldehyde production in the purpald assay. Enzymes with improvedN-demethylation activity were verified by direct measurement ofN-demethylated product (noroxymorphone, for example) formation in theculture medium by LC-MS.

Example 16: Modification of Nor-Opioid Compounds to Generate Nal-Opioids

Nor-opioid molecules can be modified at the exposed nitrogen to generatenal-opioids (see FIG. 25), an important class of pharmacotherapies forcombating opioid addiction and opioid-associated side effects. Toidentify enzymes capable of modifying nor-opioid molecules, sequencesfrom Table 5 were subjected to codon optimization for expression in S.cerevisiae, and ordered as synthetic genes (from Integrated DNATechnologies). Codon optimized sequences were cloned into expressionvectors pA24, pA25, or pA26 (or similar vectors), shown in FIG. 28,which harbor promoter sequences of varying strength, by gap repair inthe Cen.PK2 yeast host strain, according to standard molecular biologyprocedures. Individual colonies were isolated and verified by PCR andsequencing (ELIM biopharmaceuticals).

Strains expressing putative modifying enzymes were then tested for basallevels of activity on the various substrates listed in FIG. 25. Todetect activity, cell cultures were grown in selective medium (asdescribed above), lysed by glass bead disruption, and incubated withsubstrates in the presence of redox molecules and other cofactors (NADH,NADPH, and iron, for example). N-methylation of substrates was testedusing S-adenosylmethionine (SAM) as the cosubstrate, and then additionalmodifying activity of enzymes was tested using SAM analogues (see“Cosubstrates” in FIG. 25). Modification of BIA substrates was detectedvia LC-MS of experimental and control samples.

To identify engineered enzymes with improved BIA modifying activity,sequences encoding enzymes listed in Table 5 were subjected to randommutagenesis and then screened via a high-throughput colorimetric assay.Initial libraries were generated by error-prone PCR using Mutazyme II(Agilent Technologies), and variants were cloned into the pA24 (orsimilar) vector by gap repair in the Cen.PK2 screening host. Strainsexpressing mutated enzymes, with either individual mutations orcombinations of mutations introduced by gene shuffling or other methods,were grown in selective medium in 96-well plate format under varyingfermentation conditions (different media components, pH, andtemperature, for example), pelleted, and lysed by glass bead disruption.To detect N-methylation activity in a high-throughput screen, lysateswere incubated with substrates (such as noroxymorphone) in the presenceof a BM3 variant with demethylating activity, and assayed forformaldehyde production in the purpald assay (for indirect measurementof methylation). In this case, formaldehyde formation can only resultfrom the activity of BM3 on a substrate that has been N-methylated by anenzyme of interest. Enzymes with improved modifying activity wereadditionally tested for activity in cell lysates using various SAManalogues as cosubstrates (see FIG. 25), and verified by directmeasurement of product formation by LC-MS. The best variant enzymes wereselected for the efficient bioconversion of substrate molecules tonal-opioid compounds.

Example 17: Demethylase Activity of BM3 Enzyme on Opioid Molecules

BM3 is a Bacillus megaterium cytochrome P450 involved in fatty acidmonooxygenation in its native host. It is also readily expressed as anactive heterologous enzyme in yeast and bacteria. BM3 has severaladvantages as a biosynthetic enzyme including that it is soluble, comeswith a fused reductase partner protein, and can readily be engineered toaccept new substrates. Several known BM3 variants have specific alaninesubstitutions which allow the rigid morphinan pentacyclic structure toaccess the active site. These variants were expressed in yeast andobserved to N-demethylate thebaine to northebaine.

Specifically, BM3 variants 4H9, 7A1, and 8F11 (listed in Table 6) wereintegrated into the genomes of individual yeast strains (CEN.PK2) andincubated in citric acid-phosphate buffer (pH 5.0, 6.0, 7.0) andTris-HCl buffer (pH 7.5, 8.0, 8.5) with 100 μM thebaine for 20 hours. Agenetic construct which was identical except for the exclusion of theBM3 open reading frame was integrated to generate a no-enzyme controlstrain. The cells expressing BM3 produced northebaine at all tested pHlevels above 7.0. The northebaine generated by the yeast strains wasquantified by liquid chromatography mass spectrometry. The mass spectrumof northebaine (m/z 298) lacked the m/z 58 product ion consistent with ademethylated nitrogen (see FIG. 27C).

FIG. 27A, FIG. 27B and FIG. 27C illustrate the functional expression ofBM3 variants, in accordance with embodiments of the invention. FIG. 27Ashows the reaction mediated by the BM3 N-demethylase enzyme. FIG. 27Billustrates the functional expression of BM3 variants with thebaineN-demethylase activity in yeast. BM3 variants 4H9, 7A1, and 8F11 wereintegrated into the genomes of individual yeast strains (CEN.PK2) andincubated in citric acid-phosphate buffer (pH 5.0, 6.0, 7.0) andTris-HCl buffer (pH 7.5, 8.0, 8.5) with 100 μM thebaine for 20 hours. Agenetic construct which was identical except for the exclusion of theBM3 open reading frame was integrated to generate a no-enzyme controlstrain. The cells expressing BM3 produced northebaine at all tested pHlevels above 7.0. The northebaine generated by the yeast strains wasquantified by liquid chromatography mass spectrometry. The mass spectrumof northebaine (m/z 298) lacked the m/z 58 product ion consistent with ademethylated nitrogen (see FIG. 27C).

Example 18: Biological Production of O-Demethylated Opioid Molecules

Enzymes described in Example 14 and listed in Table 3, that displayedO-demethylase activity on BIA molecules (such as those listed in Table2), were incorporated into a microbial strain (either Saccharomycescerevisiae or Escherichia coli) which biosynthesizes morphinan alkaloidsde novo. The complete BIA biosynthetic pathway uses tyrosine produced bythe host cell and/or supplemented in the culture medium. Two moleculesof tyrosine are modified and condensed to form the firstbenzylisoquinoline structure which may be either norcoclaurine ornorlaudanosoline. The benzylisoquinoline is further modified to form(S)-reticuline and then stereochemically inverted by the activity of anepimerase enzyme to yield (R)-reticuline. (R)-reticuline undergoes acarbon-carbon coupling reaction to form the first promorphinan,salutaridine, and is further modified before undergoing an oxygen-carboncoupling reaction to arrive at the first morphinan alkaloid structure,thebaine (see FIG. 26). Table 2 lists enzymes and activities in thecomplete pathway.

FIG. 26 illustrates a biosynthesis scheme in a microbial cell, inaccordance with embodiments of the invention. Tyrosine producedendogenously by the cell and/or supplied in the culture medium isconverted to oxycodone (broken arrows represent multiple enzymaticsteps). The oxycodone is then 3-O-demethylated to oxymorphone andN-demethylated to noroxymorphone. Finally, an N-methyltransferaseaccepts allyl and cyclopropylmethyl carbon moieties from SAM analoguesto produce naloxone and naltrexone, respectively.

To detect O-demethylase activity in strains producing morphinan alkaloidmolecules (see FIG. 26), cells expressing candidate enzymes, either fromplasmid vectors or chromosomally-integrated cassettes, were propagatedby fermentation and cell supernatants were collected to analyze thetotal opioid profile (as described above). O-demethylation of opioidmolecules in strains harboring the complete BIA pathway was detected byLC-MS (as described above). Specifically, the conversion of oxycodone tooxymorphone was detected. To detect O-demethylation activity viabiocatalysis, strains were cultured in selective medium and then lysedby glass bead disruption. Cell lysates were supplied exogenously withopioid substrates (see FIG. 23), and other cofactors necessary forenzyme function. O-demethylation of opioid molecules was detected byLC-MS.

Example 19: Biological Production of N-Demethylated Opioid Molecules

Enzymes described in Example 15 and listed in Table 4, that displayedN-demethylase activity on BIA molecules (such as those listed in Table2), were incorporated into a microbial strain (either Saccharomycescerevisiae or Escherichia coli) which biosynthesizes morphinan alkaloidsde novo. The complete BIA biosynthetic pathway uses tyrosine produced bythe host cell and/or supplemented in the culture medium. Two moleculesof tyrosine are modified and condensed to form the firstbenzylisoquinoline structure which may be either norcoclaurine ornorlaudanosoline. The benzylisoquinoline is further modified to form(S)-reticuline and then stereochemically inverted by the activity of anepimerase enzyme to yield (R)-reticuline. (R)-reticuline undergoes acarbon-carbon coupling reaction to form the first promorphinan,salutaridine, and is further modified before undergoing an oxygen-carboncoupling reaction to arrive at the first morphinan alkaloid structure,thebaine (see FIG. 26). Table 2 lists enzymes and activities in thecomplete pathway.

To detect N-demethylase activity in strains producing morphinan alkaloidmolecules (see FIG. 26), cells expressing candidate enzymes, either fromplasmid vectors or chromosomally-integrated cassettes, were propagatedby fermentation and cell supernatants were collected to analyze thetotal opioid profile (as described above). N-demethylation of opioidmolecules in strains harboring the complete BIA pathway was detected byLC-MS (as described above). Specifically, the conversion of oxymorphoneto noroxymorphone was detected. To detect N-demethylation activity viabiocatalysis, strains were cultured in selective medium and then lysedby glass bead disruption. Cell lysates were supplied exogenously withopioid substrates (see FIG. 24), and other cofactors necessary forenzyme function. N-demethylation of opioid molecules was detected byLC-MS.

Example 20: Biological Production of Nal-Opioid Compounds

Enzymes described in Example 16 and listed in Table 5, that displayedN-methylase activity on BIA molecules (such as those listed in Table 2),were incorporated into a microbial strain (either Saccharomycescerevisiae or Escherichia coli) which biosynthesizes morphinan alkaloidsde novo. FIG. 26 shows an example of the complete reaction scheme fromthe precursor molecule thebaine to the final nal-opioid compoundsnaloxone and naltrexone. These strains additionally express enzymes fromExamples 1 and 2 and Tables 1 and 2, that are responsible for generatingnor-opioid compounds from the complete BIA pathway. N-methylase enzymeswere also expressed in a microbial strain (either Cen.PK2 for S.cerevisiae or BL21 for E. coli, for example) lacking the biosyntheticpathway, to generate a strain that is capable of biocatalysis of severaldifferent exogenously-supplied substrate molecules. The complete BIAbiosynthetic pathway uses tyrosine produced by the host cell and/orsupplemented in the culture medium. Two molecules of tyrosine aremodified and condensed to form the first benzylisoquinoline structurewhich may be either norcoclaurine or norlaudanosoline. Thebenzylisoquinoline is further modified to form (S)-reticuline and thenstereochemically inverted by the activity of an epimerase enzyme toyield (R)-reticuline. (R)-reticuline undergoes a carbon-carbon couplingreaction to form the first promorphinan, salutaridine, and is furthermodified before undergoing an oxygen-carbon coupling reaction to arriveat the first morphinan alkaloid structure, thebaine (see FIG. 26). Table2 lists enzymes and activities in the complete pathway.

To detect N-modifying activity in strains with the complete BIA pathwayto nor-opioids (see FIG. 26), cells expressing candidate enzymes werepropagated by fermentation (as described above) and incubated with SAMor SAM analogs, such as those listed in FIG. 25. Enzymatic modificationof nor-opioid or other BIA molecules in strains harboring the completeBIA pathway was detected in supernatants by LC-MS (as described above).To detect N-modifying activity via biocatalysis, strains were culturedin selective medium and then lysed by glass bead disruption. Celllysates were supplied exogenously with SAM or SAM analogs, and othercofactors necessary for enzyme function. Specifically, the conversion ofnoroxymorphone to naloxone and naltrexone (using the SAM analogsallyl-SAM or cyclopropane-SAM, as shown in FIG. 25) was detected.Modification of nor-opioid or other BIA molecules was detected by LC-MS.To detect N-modifying activity by biocatalysis in a strain that does nothave the complete BIA pathway, Cen.PK2 strains expressing enzymesdescribed in Example 16 were grown in selective medium and lysed byglass bead disruption. Cell lysates were supplied exogenously with SAMor SAM analogs, cofactors necessary for enzyme function, and nor-opioidmolecules such as those listed in FIG. 25 and Table 2. Modification ofthese compounds was detected by LC-MS.

Example 21: O-Demethylase Activity of CODM on Opioid Molecules

FIG. 29 illustrates the functional expression of CODM, in accordancewith embodiments of the invention. In particular, FIG. 29 illustratesthe functional expression of CODM with oxycodone 3-O-demethylaseactivity in yeast. The yeast codon-optimized CODM gene was integratedinto the genome of yeast strain W303 and cultured in synthetic completemedia for 16 hours. The parent W303 strain was also cultured insynthetic complete media for 16 hours as a no-enzyme control. The cellswere pelleted and washed with 1 mL breaking buffer (100 mM Tris-HCl pH7.5, 10% glycerol, 14 mM 2-mercaptoethanol, lx protease inhibitor).Cells were resuspended in 200 μL breaking buffer and lysed by glass beaddisruption. The crude cell lysates were incubated with 10 mM ascorbicacid, 0.5 mM iron(II) sulfate, 0.1 mM oxycodone as substrate and 10 mM2-oxoglutarate as cosubstrate in a total volume of 100 μL. 4 mM DTT wasalso added as a reducing agent to keep iron in the Fe²⁺ state. Thereaction was incubated at 30° C. for 3 h and quenched by diluting it 1:1in ethanol with 0.1% acetic acid. The oxymorphone generated by the yeaststrain expressing CODM was detected by LC-MS. The mass-charge ratio (m/z302), retention time, and mass spectrum of oxymorphone produced by theyeast strain matched that of a purchased oxymorphone standard (see FIG.29).

TABLE 2 Enzyme list Source Genbank Enzyme Abbrev Catalyzed Reactionsorganisms # 3-deoxy-d-arabinose- ARO4, erythrose-4-phosphate +Saccharomyces CAA85212.1 heptulosonate-7- DHAP PEP → DHAP (EC 2.5.1.54)cerevisiae phosphate synthase synthase Chorismate mutase ARO7 chorismate→ prephenate Saccharomyces NP_015385.1 (EC 5.4.99.5) cerevisiaePhenylpyruvate ARO10 hydroxyphenylpyruvate → Saccharomyces NP_010668.3decarboxylase 4HPA (EC 4.1.1.80) cerevisiae Aromatic ARO9hydroxyphenylpyruvate + Saccharomyces AEC14313.1 aminotransferaseglutamate → tyrosine + cerevisiae alpha-ketogluterate (EC 2.6.1.57)Transketolase TKL1 fructose-6-phosphate + Saccharomyces NP_015399.1glyceraldehyde-3- cerevisiae phosphate ↔ xylulose-5- phosphate +erythrose-4- phosphate (EC 2.2.1.1) Glucose-6-phosphate ZWF1glucose-6-phosphate → 6- Saccharomyces CAA96146.1 dehydrogenasephosphogluconolactone cerevisiae (EC 1.1.1.49) Alcohol dehydrogenaseADH2-7, 4HPA → tyrosol (EC Saccharomyces NP_014032.1, SFA1 1.1.1.90)cerevisiae AAT93007.1, NP_011258.2, NP_009703.3, NP_014051.3,NP_010030.1, NP_010113.1 Aldehyde oxidase ALD2-6 4HPA → SaccharomycesNP_013893.1, hydroxyphenylacetic acid cerevisiae NP_013892.1, (EC1.2.1.39) NP_015019.1, NP_010996.2, NP_015264.1 Tyrosinase TYR tyrosine→ L-DOPA, L- Ralstonia NP_518458.1, DOPA → dopaquinone (EC solanacearum,AJ223816, 1.14.18.1) Agaricus bisporus Tyrosine hydroxylase TyrHtyrosine → L-DOPA (EC Homo NM012740, 1.14.16.2) sapiens, NM000240,Rattus norvegicus, Mus musculus GTP cyclohydrolase FOL2 GTP →dihydroneopterin Saccharomyces CAA97297.1, triphosphate (EC 3.5.4.16)cerevisiae, NP_001019195.1, Homo NP_032128.1 sapiens, Mus musculus6-pyruvoyl PTPS dihydroneopterin Rattus AAH59140.1, tetrahydrobiopterintriphosphate → PTP (EC norvegicus, BAA04224.1, (PIP) synthase 4.2.3.12)Homo AAH29013.1 sapiens, Mus musculus Sepiapterin reductase SepR PTP →BH4 (EC 1.1.1.153) Rattus NP_062054.1, norvegicus, NP_003115.1, HomoNP_035597.2 sapiens, Mus musculus 4a- PCD 4a- Rattus NP_001007602.1,hydroxytetrahydrobiopterin hydroxytetrahydrobiopterin norvegicus,AAB25581.1, (pterin-4α- → H2O + quinoid Homo NP_079549.1 carbinolannine)dihydropteridine (EC sapiens, dehydratase 4.2.1.96) Mus musculus QuinoidQDHPR quinoid dihydropteridine Rattus AAH72536.1, dihydropteridine → BH4(EC 1.5.1.34) norvegicus, NP_000311.2, reductase Homo AAH02107.1sapiens, Mus musculus L-DOPA decarboxylase DODC L-DOPA → dopamine (ECPseudomonas AE015451.1, 4.1.1.28) putida, NP_001257782.1 Rattusnorvegicus Tyrosine/DOPA TYDC L-DOPA → dopamine (EC Papaver AAA97535.1,decarboxylase 4.1.1.28) somniferum CAB56038.1 Monoamine oxidase MAOdopamine → 3,4-DHPA (EC E. coli, Homo J03792, 1.4.3.4) sapiens, D2367,Micrococcus AB010716.1 luteus Dihydrofolate DHFR 7,8-Dihydrobiopterin →Rattus AF318150.1 reductase 5,6,7,8- norvegicus, Tetrahydrobiopterin(BH4) Homo EC 1.5.1.3 sapiens Norcoclaurine 6-O- 6OMT Norcoclaurine P.AY268894 methyltransferase → coclaurine somniferum AY610507Norlaudanosoline T. flavum D29811 → 3′hydroxycoclaurine Coptis EC2.1.1.128 japonica* Coclaurine-N- CNMT Coclaurine → N- P. AY217336methyltransferase methylcoclaurine somniferum AY6105083'hydroxycoclaurine T. flavum AB061863 → 3'-hydroxy-N- Coptismethylcoclaurine japonica* EC 2.1.1.140 4'-O-methyltransferase 4'OMT3'-hydroxy-N- P. AY217333, methylcoclaurine somniferum AY217334 →Reticuline EC 2.1.1.116 T. flavum AY610510 Coptis D29812 japonica*Norcoclaurine synthase NCS 4HPA + dopamine → S- Coptis BAF45337.1,norcoclaurine (EC 4.2.1.78) japonica, ACI45396.1, 3,4-DHPA + dopamine →S- Papaver AC090258.1, norlaudanosoline somniferum, AC090247.1, PapverAEB71889.1 bracteatum, Thalicitum flavum, Corydalis saxicola CytochromeP450 80B1 CYP80B1 N-methylcoclaurine → 3'- P. AAF61400.1,hydroxy-N-methylcoclaurine somniferum, AAC39453.1, E. californica,AAU20767.1 T. flavum Cheilanthifoline synthase CFS Scoulerine P.GU325749 → cheilanthifoline somniferum AB434654 EC 1.14.21.2 E.californica EF451152 A. mexicana Stylopine synthase STS CheilanthifolineP. GU325750 → stylopine somniferum AB126257 EC 1.14.21.1 E. californicaEF451151 A. mexicana Tetrahydroprotoberberine-N- TNMT Stylopine → cis-N-P. DQ028579 methyltransferase methylstylopine somniferum EU882977 EC2.1.1.122 E. californica EU882994 P. HQ116698 bracteatum A. mexicanaCis-N-methylstylopine 14- MSH cis-N-methylstylopine P. KC154003hydroxylase → protopine somniferum EC 1.14.13.37 Protopine-6-hydroxylaseP6H Protopine → 6- E. californica AB598834 hydroxyprotopine P. AGC92397EC 1.14.13.55 somniferum Dihydrobenzophenanthridine DBOXDihydrosanguinarine P. [not in oxidase → sanguinarine EC somniferumgenbank] 1.5.3.12 (S)-tetrahydroprotoberberine STOX(S)-tetrahydroberberine + 2 Berberis HQ116697, oxidase O₂ = berberine +2 H₂O₂ wilsonae, AB564543 EC 1.3.3.8 Coptis japonica, Berberis spp,Coptis spp S-adenosyl-L-nnethionine:(S)- S9OMT S-adenosyl-L-methionine +Thalictrum AY610512, scoulerine 9-O- (S)-scoulerine = S-adenosyl- flavumD29809, methyltransferase L-homocysteine + (S)- subsp. EU980450,tetrahydrocolunnbamine glaucum, JN185323 EC 2.1.1.117 Coptis japonica,Coptis chinensis, Papaver somniferum, Thalictrum spp, Coptis spp,Papaver spp (S)- CAS (S)-tetrahydrocolumbannine + Thalictrum AY610513,tetrahydrocolunnbannine, NAD NADPH + H+ + O2 = (S)- flavum AB026122, PH:oxygen oxidoreductase canadine + NADP+ + 2 H2O subsp. AB374407,(methylenedioxy-bridge- EC 1.14.21.5 glaucum, AB374408 forming), alsoknown as (S)- Coptis canadine synthase japonica, Thalictrum spp, Coptisspp (S)-reticuline: oxygen BBE (S)-reticuline + O2 =(S)- PapaverAF025430, oxidoreductase (methylene- scoulerine + H2O2 somniferum,EU881889, bridge-forming), also known EC 1.21.3.3 Argemone EU881890, asberberine bridge enzyme mexicana, S65550 Eschscholzia AF005655,californica, AF049347, Berberis AY610511, stolonifera, AB747097Thalictrum flavum subsp. glaucum, Coptis japonica, Papaver spp,Eschscholzia spp, Berberis spp, Thalictrum spp, Coptis spp NADPH:hemoprotein ATR1, NADPH + H+ + n oxidized Arabidopsis CAB58576.1,oxidoreductase, also known CPR hemoprotein = NADP+ + thaliana,CAB58575.1, as cytochrome P450 n reduced hemoprotein EC EschscholziaAAC05021.1, reductase 1.6.2.4 californica, AAC05022.1, Papaver NM118585,somniferum, many Homo others sapiens, (Ref Saccharomyces PMIDcerevisiae, 19931102) Papaver bracteatum, Papaver spp, all plantssalutaridinol: NADP+ 7- SaIR salutaridinol + NADP+ = Papaver DQ316261,oxidoreductase, also known salutaridine + NADPH + H+ somniferum,EF184229 as salutaridine reductase EC 1.1.1.248 Papaver (Ref bracteatum,PMID Papaver spp 22424601) Chelidonium majus acetyl-CoA: salutaridinol7-O- SalAT acetyl-CoA + salutaridinol = Papaver AF339913,acetyltransferase, also known CoA + 7-O- somniferum, FJ200355, assalutaridinol 7-O- acetylsalutaridinol Papaver FJ200358,acetyltransferase EC 2.3.1.150 bracteatum, FJ200356, Papaver JQ659008orientale, Papaver spp (R)-reticuline, NADPH: oxygen SalSyn(R)-reticuline + NADPH + Papaver EF451150 oxidoreductase (C-C phenol-H+ + O2 = salutaridine + somniferum, (Ref coupling), also known asNADP+ + 2 H2O Papaver spp PMID salutaridine synthase EC 1.14.21.4Chelidonium 22424601) majus 1-benzylisoquinoline alkaloid CYP-COR(S)-reticuline -> (R)-reticuline Papaver P0DKI7.1, epimerase (cytochromeP450 or DRS- (S)-1-benzylisoquinoline- bracteatum, AKO60175.1, 82Y1-likecodeinone DRR >(R)-1-benzylisoquinoline Papaver AKO60180.1,reductase-like) EC 1.5.1.27 somniferum, AKO60179.1, Papaver AKO60175.1setigerum, Chelidonium majus Cytochrome P450, family 2, CYP2D6Promiscuous oxidase, can Homo BC067432 subfamily D, polypeptide 6perform sapiens (R)-reticuline + NADPH + H+ + O2 = salutaridine +NADP+ + 2 H2O among other reactions EC 1.14.14.1 Thebaine 6-Odemethylase T6ODM thebaine → 

 neopinone Papaver GQ500139.1 EC somniferium, 1.14.11.31 Papaver spp.Codeinone reductase COR codeinone → 

 codeine EC Papaver AF108432.1 1.1.1.247, somniferium, AF108433.1neopinone → 

 neopine Papaver AF108434.1 spp. AF108435.1 Codeine O-demethylase CODMcodeine → 

 morphine EC Papaver GQ500141.1 1.14.11.32, somniferium, neopine → 

 neomorphine Papaver spp. Morphine dehydrogenase morA morphine → 

 orphinone Pseudomonas M94775.1 1.1.1.218, EC putida codeinone → 

 codeine EC 1.1.1.247 Morphinone reductase morB codeinone -hydrocodonePseudomonas U37350.1 morphinone putida

 

 hydromorphoneEC 1.3.1.- Reticuline N- RNMT reticuline→tembetarinePapaver KX369612.1 methyltransferase somniferum, Papaver spp. Papaverine7-O-demethylase P7OMT papaverine→pacodine Papaver K1159979.1 somniferum,Papaver spp. 3-O-demethylase 3ODM oxycodone→oxymorphone Papaverhydrocodone→hydromorphone somniferum, dihydrocodeine→dihydromorphinePapaver 14-hydroxycodeine→14- bracteatum, hydroxymorphine Papavercodeinone→morphinone rhoeas, 14-hydroxycodeinone→14- Papaverhydroxymorphinone spp. N-demethylase NDM Codeine→Norcodeine BacillusMorphine→Normorphine megaterium, Oxycodone→Noroxycodone HomoOxymorphone→Noroxymorphone sapiens, Thebaine→Northebaine PapaverOripavine→Nororipavine somniferum, Hydrocodone→Norhydrocodone PapaverHydromorphone→Norhydromorphone spp., Dihydrocodeine→NordihydrocodeineChelidonium Dihydromorphine→Nordihydromorphine majus,14-hydroxycodeine→Nor-14- Stylophorum hydroxycodeine diphyllum,14-hydroxymorphine→Nor- Nigella 14-hydroxymorphine sativa, Hydrastiscanadensis, Glaucium flavum, Codeinone→Norcodeinone EschscholziaMorphinone→Normorphinone californica, 14-hydroxycodeinone→Nor-Menispermum 14-hydroxycodeinone canadense, 14- Papaverhydroxymorphinone→Nor- bracteatum 14-hydroxymorphinoneN-methyltransferase NMT Norcodeine→codeine Papaver Normorphine→morphinespp., Noroxycodone→oxycodone Chelidonium Noroxymorphone→noroxymorphonemajus, Northebaine→thebaine Thalictrum Nororipayine→oripayine flavum,Norhydrocodone→hydrocodone Coptis Norhydromorphone→ japonica,Hydromorphone Papaver Nordihydrocodeine→ somniferum, DihydrocodeineEschscholzia Nordihydromorphine→ californica, Dihydromorphine PapaverNor-14-hydroxycodeine→ bracteatum, 14-hydroxycodeine ArgenomeNor-14-hydroxymorphine→ mexicana, 14-hydroxymorphine GlauciumNorcodeineone→ flavum, Codeineone Sanguinaria Normorphinone→ canadensis,Morphinone Corydalis Nor-14-hydroxy-codeinone→ chelanthifolia,14-hydroxycodeinone Nigella Nor-14-hydroxy- sativa, morphinone→14-Jeffersonia hydroxymorphinone diphylla, Berberis thunbergii, Mahoniaaquifolium, Menispermum canadense, Tinospora cordifolia, Cissampelosmucronata, Cocculus trilobus N-allyltransferase NAT Norcodeine→N-allyl-Papaver norcodeine spp., Normorphine→N-allyl- Chelidonium normorphinemajus, Noroxycodone→N-allyl- Thalictrum noroxycodone flavum,Noroxymorphone→N-allyl- Coptis nornoroxymorphone japonica,Northebaine→N-allyl- Papaver northebaine somniferum,Nororipavine→N-allyl- Eschscholzia nororipavine californica,Norhydrocodone→N-allyl- Papaver norhydrocodone bracteatum,Norhydromorphone→N- Argenome allyl-norhydromorphone mexicana,Nordihydrocodeine→N-allyl- Glaucium nordihydrocodeine flavum,Nordihydromorphine→N- Sanguinaria allyl-nordihydromorphine canadensis,Nor-14-hydroxycodeine→N- Corydalis allyl-nor-14-hydroxycodeinechelanthifolia, Nor-14-hydroxymorphine→ Nigella N-allyl-nor-14- sativa,hydroxymorphine Jeffersonia Norcodeineone→N-allyl- diphylla,norcodeineone Berberis Normorphinone→N-allyl- thunbergii, normorphinoneMahonia Nor-14-hydroxy-codeinone→ aquifolium, N-allyl-nor-14-Menispermum hydroxycodeinone Nor-14-hydroxy- canadense, morphinone→N-allyl-nor- Tinospora 14-hydroxymorphinone cordifolia, Cissampelosmucronata, Cocculus trilobus N- NCPMT Norcodeine→N- Papavercyclopropylmethyltransferase (Cyclopropylmethyl)norcodeine spp.,Normorphine→N- Chelidonium (Cyclopropylmethyl)normorphine majus,Noroxycodone→N- Thalictrum (Cyclopropylmethyl)noroxycodone flavum,Noroxymorphone→N- Coptis (Cyclopropylmethyl)nornoroxymorphone japonica,Northebaine→N- Papaver (Cyclopropylmethyl)northebaine somniferum,Nororipavine→N- Eschscholzia (Cyclopropylmethyl)nororipavinecalifornica, Norhydrocodone→N- Papaver (Cyclopropylmethyl)norhydrocodonebracteatum, Nordihydrocodeine→N- Argenome(Cyclopropylmethyl)nordihydrocodeine mexicana, Nordihydromorphine→N-Glaucium (Cyclopropylmethyl)nordihydromorphine flavum,Nor-14-hydroxycodeine→N- Sanguinaria (Cyclopropylmethyl)nor-14-canadensis, hydroxycodeine Corydalis Nor-14-hydroxymorphine→chelanthifolia, N-(Cyclopropylmethyl)nor- Nigella 14-hydroxymorphinesativa, Norcodeineone→N- Jeffersonia (Cyclopropylmethyl)norcodeineonediphylla, Normorphinone→N- Berberis (Cyclopropylmethyl)normorphinonethunbergii, Nor-14-hydroxy-codeinone→ Mahonia N-(Cyclopropylmethyl)nor-aquifolium, 14-hydroxycodeinone Menispermum Nor-14-hydroxy- canadense,morphinone→N- Tinospora (Cyclopropylmethyl)nor-14- cordifolia,hydroxymorphinone Cissampelos mucronata, Cocculus trilobus

TABLE 3 O-demethylase candidate enzymes SEQ ID Name Sequence NO: T60DMMEKAKLMKLGNGMEIPSVQELAKLTLAEIPSRYVCANENLLLPMGAS 16VINDHETIPVIDIENLLSPEPIIGKLELDRLHFACKEWGFFQVVNHGVDASLVDSVKSEIQGFFNLSMDEKTKYEQEDGDVEGFGQGFIESEDQTLDWADIFMMFTLPLHLRKPHLFSKLPVPLRETIESYSSEMKKLSMVLFNKMEKALQVQAAEIKGMSEVFIDGTQAMRMNYYPPCPQPNLAIGLTSHSDFGGLTILLQINEVEGLQIKREGTWISVKPLPNAFVVNVGDILEIMTNGIYHSVDHRAVVNSTNERLSIATFHDPSLESVIGPISSLITPETPALFKSGSTYGDLVEECKTRKLDGKSFLDSMRI CODMMETPILIKLGNGLSIPSVQELAKLTLAEIPSRYTCTGESPLNNIGAS 17VTDDETVPVIDLQNLLSPEPVVGKLELDKLHSACKEWGFFQLVNHGVDALLMDNIKSEIKGFFNLPMNEKTKYGQQDGDFEGFGQPYIESEDQRLDWTEVFSMLSLPLHLRKPHLFPELPLPFRETLESYLSKMKKLSTVVFEMLEKSLQLVEIKGMTDLFEDGLQTMRMNYYPPCPRPELVLGLTSHSDFSGLTILLQLNEVEGLQIRKEERWISIKPLPDAFIVNVGDILEIMTNGIYRSVEHRAVVNSTKERLSIATFHDSKLESEIGPISSLVTPETPALFKRGRYEDILKENLSRKLDGKSFLDYMRM PsP7ODMMEKAKLMKLGNGLSIPSVQELAELTFAEVPSRYVCTNDENLLLMTM 18GASEIDDETVPVIDLQNLLSPEPAIGKSELDWLHYSCKEWGFFQLVNHGVDALLVDHVKSEIHSFFNLPLNEKTKYGQRDGDVEGFGQAFLVSENQKLDWADMFFINTLPLHLRKPHLFPNLPLPLRETIESYSSEMKKLSMVLFEMMGKAIEVIDIKEAITEMFEDGMQSMRMNYYPPCPQPERVIGITPHSDFDGLTILLQLNEVEGLQIRKEDKWISIKPLPDAFIVNVGDIWEIMTNGVHRSVDHRGVFNSTKERLSIATFHSPKLELEIGPISSLIRPETPAVFKSAGRFEDLLKEGLSRKLDGKSFLDCMRM PsoDIOX1MEKAKLMKLGNGMEIPSVQELAKLTLAEIPSRYVCANENLLLPMGAS 19VINDHETIPVIDIENLLSPEPIIGKLELDRLHFACKEWGFFQVVNHGVDASLVDSVKSEIQGFFNLSMDEKTKYEQEDGDVEGFGQGFIESEDQTLDWADIFMMFTLPLHLRKPHLFSKLPVPLRETIESYSSEMKKLSMVLFNKMEKALQVQAAEIKGMSEVFIDGTQAMRMNYYPPCPQPNLAIGLTSHSDFGGLTILLQINEVEGLQIKREGTWISVKPLPNAFVVNVGDIL EIMTNGIYHSVD PsoDIOX2METAKLMKLGNGMSIPSVQELAKLTLAEIPSRYICTVENLQLPVGAS 20VIDDHETVPVIDIENLISSEPVTEKLELDRLHSACKEWGFFQVVNHGVDTSLVDNVKSDIQGFFNLSMNEKIKYGQKDGDVEGFGQAFVASEDQTLDWADIFMILTLPLHLRKPHLFSKLPLPLRETIESYSSEMKKLSMVLFEKMEKALQVQAVEIKEISEVFKDMTQVMRMNYYPPCPQPELAIGLTPHSDFGGLTILLQLNEVEGLQIKNEGRWISVKPLPNAFVVNVGDVLEIMTNGMYRSVDHRAVVNSTKERLSIATFHDPNLESEIGPISSLITPNTPALFRSGSTYGELVEEFHSRKLDGKSFLDSMRM PbrDIOX2METPKSIKLGGSLLVPSVQELAQQSFAEVPARYVRDDLEPLTDLSGV 21SMIDQTIPVIDLQKLQSPVPIIRELESEKLHSACKEWGFFQVVNHGVDILLVEKTKSEIKDFFNLPMDEKKKFWQEEGDIQGFGQAFVQSEDQKLDWADIFLMVTLPRHTRNPRLFPKLPLPLRNTMDSYSSKLSKLASTLIEMMGKALHMETSVLAELFEDGRQTMRINYYPPCPQPKDVIGLTPHSDGGGLTILLQLNEVDGLQIRKEKIWIPIKPLPNAFVVNIGNILEIMTNGIYRSVEHRATIHSTKERLSVAAFHNPKVGVEIGPIVSMITPESPALFRTIEYDDYGKKYFSRKLDGKSSLDFMRIGEGDEENKAT PbrDIOX3METPKLIKLGGSLLVPSVLELTKQSPAEVPARYIRNDLEPMTDLSSA 22SLTDQTIPVIDLQNLLSPEPELELEKLHSGCKEWGFFQVMNHGVDILLVEKVKSEIQGFFNLPIDEKNKFWQEEGDLEGYGKAFVHSEDEKLDWADMFFILTQPQYMRKPRVFPKLPLRLRETIESYSLELSKLGLTLLDLMGKALQIETGVMSELFEDGRQTMRMNYYPPCPQPEHVIGLTPHSDGGALTILLQLNQVDGLQIRKEEIWVPIKPLPNAFVVNIGDILEIMSNGVYRSVEHRATINSSKERLSVAIFQSPKHGTEIGPILSMITPEAPALFKTIPYEDYLRKFFSRKLGGKSFVDSMRIGESDEDNNTA PbrDIOX4METQKQENFGASLSVPNVQELAKQSPEQVPDRYIRSDQDSSTNISCP 23SMTDQIPVIDLQSLLSPDPIIGELELERLHSACKEWGFFQVVNHGVDNLLVEKVKSEIQGFFNLPMDEKKKFWQEEGDFEGFGQAFVFSEDQKLDWGDVFFILTQPQHMRKPRLFPKLPLPFRKTIESYSLETNKLSMTLLELMEKALKIETGVMTELFEGGIQRMRMTYYPPCPQPKHVIGLTPHSDPDALTILLQLNEVDGLQIRKEKIWVPIKPLSNAFVVNIGDILEIMSNGIYRSVEHRATVNSTKERLSVATFHSPRKDTEIGPILITPETPALFRTSGFEDYFRKFFAHKLNGKSFLSSIRIGETDEGNNAT PbrDIOX5MEAPKLIMLGGSLFVPSVQELAKQSLAEVPVRYVRDDQDTLGNNINI 24TPMSMIDQSIPVIDLEKLLSPEPIVGELELERLHSACKEWGFFQVVNHGVDSLLVEKVKSEIEGFFKLPMDEKTKFWQEEGDIEGFGQVFVHSQDQKLDWGDMFLMQTLPRHTRKPRLFPNLPLPLRQTIESYSSELSKLVLTLVDLMGKALQMESGVLTELFENGIQRMRMNYYPPCPQPEQVIGLTPHSDVGGLTILLQLNEVDGLQIKKDKVWVPIKPLANAFVVNVGDALEIMSNGIYRSVEHRATINSTKERLSIATFHNPRADREIGPIPSMISPETPALFKTTGYEEYFKKFFSRKLEGKSFLDSLRIREGDEHCGRLDVKG PCN PhrDIOX6MEIPNPIKIGSSLLVPSVQELAKQSFAEVPARYIRNDVDPLITKLSD 25VSLIDQTVPVIDLQKLLSPEPIVGELELERLHSACKEWGFFQVVNHGVDNLLVEKVKSEIQGFFNLPMEEKKKFWQEEGDFEGFGQMFVQSEEQKLDWGDMFFILTQPQHMRKPRLFSKLPLPLRETIESYSLELIKLGLTIIKLMEKALQIDAGVMAELFEDGIHTMRMNYYPPCPQPEHVIGLTPHSDGGGLTILLQLNEVDGLQIRRENIWVPIKPLPNAFVVNIGDILEILSNGIYRSVEHRSTVNATKERLSVATFQNPKQESVIGPNMITPERPALFRKIVYKDYMKKLFSRKLDGKSFLDSLRIGEGDERP PbrDIOX8METLKTVKPGGSLFIPNGQELAKQSLEEVYVGNDQDTMLLIGQTIPV 26IDLQKLLSPEPITGDMELDKLHSACKEWGFFQVVNHGVDILLVEKVKSEVHDFFNIPMDEKKPFWQEEGDLEGFGQVFITSEDQQLDWGDMFFMVTLPKHMRKPRLFLKLPLPLRETIESYSLKLSKLGVTLVELMGKALQMEDRIMSELFDDGRQTMRMNYYPPCPQPEQVIGLTPHSDPGGLTILLELNEVNGLIRKENIWVPIIPLPNAFIVNIGDILEIMSNGIYHSVEHRATINSTKERLSVAMFNSPKVDTEIGPIHSMITPETPALFRTIGYDE YLKIFFSRKLDGKSLLESMKIPbrDIOX10 MEAPKLIMLGGSLFVPSVQELAKQSLAEVPVRYVRDDQDTLGNNINI 27TPMSMIDQSIPVIDLEKLLSPEPIVGELELERLHSACKEWGFFQVVNHGVDSLLVEKVKSEIEGFFELPVDEKKKFWQEEGDIEGFGQIFVHSEDQKLDWADMFYMLTLPPNMRKPRLFPNLPLPLRQTIDSYSSELSKLVLTLVDLMGKALQMESGVLTELFENGIQRMRMNYYPPCPQPEQVIGLTPHSDVGGLTILLQLNEVDGLQIKKDKIWVPIKPLRNAFVVNVGDALEIMSNGIYRSVEHRATINSTKERLSIATFHNPRADREIGPIPSMISPETPALFKTTGYEEYFKKFFSRKLEGKSFLDSLRIGEGDEHCGRLXVKG XCN PbrDIOX11METPKLMKLGGSLFVPSVQELAKQSLAEVPARYVRDDRDMVGNIINV 28TPMSMIDQSIPVIDLEKLLSPDLIVGELELERLHSACKEWGFFQVVNHGVDSLLVEKVKSEIEGFFELPMDEKKKFWQEEGDAEGFAQFFVQSEDQKLDYSGDMFFMLNLPQHMRKPRLFLKLPLPLRETIESYSLKLSKLGVTLVELMGKALQMEDRIMSELFDDGRQTMRMNYYPPCPQPEQVIGLTPHSDPGGLTILLELNEVNGLIRKENIWVPIIPLPNAFIVNIGDILEIMSNGIYHSVEHRATINSTKERLSVAMFNSPKVDTEIGPIHSMITPETPALFRTIGYDEYLKIFFSRKLDGKSLLESMKI PbrDIOX13METPKLRDFGSFLPVPSVQELAKQVLTEIPPRYIRTDLEALNKLSCA 29SNTDQTVPIIDMQCLLSAEPEMELEKLHSACKEWGFFRVVNHGVDNLESVKSEIESFLNLPVNAKNKYGQKQGDDQGFGSRFVLSEEQKLDWGDFFYMVTRPLYLRKPHLFPELPLPLRETIESYSSEVSKLAMALFEMMGKALKIETGVMTEIFEGGMQAMRMNYYPPCPRPDLVIGLNAHSDFGGLTILLQLNEVEGLEIRNKGEWVSVKPLANAFVVNVGDVMEILTNGIYHSVEHRATINSSKERLSVATFHYPKLETGIGPLPCMITPKTPALFGRIERYELLLRKYYARKLNGKSTLDCMRIGNGFEDDNTA PbrDIOX18MEAPKLIMLGGSLFVPSVQELAKQSLAEVPARYVRDDQDTLGNNINI 30TPMSMIDQSIPVIDLEKLLSPEPIVGELELERLHSACKEWGFFQVVNHGVDSLLVEKVKSEIEGFFELPVDEKKKFWQEEGDIEGFGQIFVHSEDQKLDWADMFYMLTLPPNMRKPRLFPNLPLPLRQTIDSYSSELSKLVLTLVDLMGKALQMESGVLTELFENGIQRMRMNYYPPCPQPEQVIGLTPHSEVGGLTILLQLNEVDGLQIRKEKIWVPIKPLSNAFIVNIGDILEIMSNGIYRSVEHRATVNSTKERLSVATFHSPRKDTEIGPILITPETPALFRTSGFEDYFRKFFAHKLNGKSFLSSIRIGETDEGNNAT PbrDIOX19MSMIDQSIPVIDLEKLLSPEPIVGELELERLHSACKEWGFFQVVNHG 31VDSLLVEKVKSEIEGFFELPVDEKKKFWQEEGDIEGFGQIFVHSEDQKLDWADMFYMLTLPPNMRKPRLFPNLPLPLRQTIDSYSSELSKLVLTLVDLMGKALQMESGVLTELFENGIQRMRMNYYPPCPQPEQVIGLTPHSDVGGLTILLQLNEVDGLQIRKEKIWVPIKPLSNAFIVNIGDILEIMSNGIYHSVEHRATINSTKERLSVAMFNSPKVDTEIGPIHSMITPETPALFRTIGYDEYLKIFFSRKLDGKSLLESMKI PbrDIOX21METPKLVKSSGSSLFLSTSVQELAKQSLPEVPARYIRTNLEPLSNVS 32GDSQSVPVIDLQKLLSSEPIIGELELDKLHSACKEWGFFQVVNHGVDNLVMEKIKTEIQGFFNLSLDEKQKFWKKEGDAEGFGQNFIESEDQKLDWGDTFGMFTLPIHMRNPRLFPELPLPLRETIESYSLDVRKLALALIGLMEKALKIKTSAMSELFEDGGQAMRMNYYPPCPQPEHVIGLTPHSDAGGLTILLQLNEVDGLQIKKDKIWVPIKPLPNAFVVNIGDILEIMTNGIYRSVEHRATINSSKERLSVAAFHSPKGDTLIGPMVSLITPETPALFRTIGYQDYMKKFMSRKLDGKSLVNSMRIGEGDEDK PbrDIOX-METPTLMKLGNGLSVPSVQELAKATLAEIPSRYICTDENLLTMGAST 33 ZSNV-TDNETVPV1DLQNLLSPEPVIGMLELDRLHSACKEWGFFQLVNHGVD 2004018ALLVDNEVQGFFNLPMDEKTKYGQKDGDDEGFGQFFVISEDQKLDWADVFYMSTLPLHSRKPHLFPELPLPLRETMESYSSEMKKLSMVLFDMMGKALQVVEIKGITELFEDGAQQIRMNYYPPCPQPELVFGLTSHSDFDGLTILLQLGEVEGLQIKKEERWISIKPLPDAFIVNVGDILEIMTNGIYRSVDHRAVVNSIKERLTIATFHDPRLEAEIGPISSLITPETPALFKRGVFEDLLKEMFLRKLDGKSFLDCMRM PrhDIOX-GNGLSVPSVQELAKQTLAEIPSRYICTDENPLITGASVVDDETVPVI 34 MVTX-NLQNLLSPEPVIGKLELDKLHSACKEWGFFQVVNHGVNDSLVDSVKS 2001522EIEGFFNLPANEKLKYGQKDGDVEGFGQHFVVSEDQKLDWADVFYMVTLPVRLRKPHLFPELPLPLRDTLDSYSSELNKLSMVLLEMMEKALKLVECKGITDFFEDGFQQMRMNYYPPCPRPELVTGLTSHSDFGGLTILLQLNDVEGLQIKKEERWISIKPLPNAFIVNIGDVLEIMSNGIYRSVDHRAVINSTKVRMSVATFHDPRLEAVIGPISSLITPETPALFKRGVF EDLLKEMFLRKLDGKSFLDCMRIPseDIOX- LMKLANGMSVPIVQELAKLTVGEIPSRYICTDGNLLTMGASVIDYET 35 JSVC-VPVIDLQNLQSREPVIEKLELDRLHSACKEWGFFQLLNHGVDASLMD 2005842NVRSEIRGFFNLPISDKMKYGQKDGDEEGFGQHFIVSEDQKLDWVDAFMMFTLPLHSRNPRLTPEFPQPLRETVESYSSEMKKLSVLLFELMEKALQVKGITEMFEDGLQSIRMNYYPPCPRPELAIGLTSHSDFDGLTILLQLNEVEGLQIKKEERWISIKPLPNAFIVNVGDVLEVMTNGIYRSVDHRAVVNSTKERLSIATFHDPELESEIGPIASLITPETPALFKRGRF KDLLKENLSTKLDGKSFLDCIRMCYP2D6 MGLEALVPLAVIVAIFLLLVDLMHRRQRWAARYSPGPLPLPGLGNLL 36HVDFQNTPYCFDQLRRRFGDVFSLQLAWTPVVVLNGLAAVREALVTHGEDTADRPPVPITQILGFGPRSQGVFLARYGPAWREQRRFSVSTLRNLGLGKKSLEQWVTEEAACLCAAFANHSGRPFRPNGLLDKAVSNVIASLTCGRRFEYDDPRFLRLLDLAQEGLKEESGFLREVLNAVPVLLHIPALAGKVLRFQKAFLTQLDELLTEHRMTWDPAQPPRDLTEAFLAEMEKAKGNPESSFNDENLRIVVADLFSAGMVTTSTTLAWGLLLMILHPDVQRRVQQEIDDVIGQVRRPEMGDQAHMPYTTAVIHEVQRFGDIVPLGVTHMTSRDIEVQGFRIPKGTTLITNLSSVLKDEAVWEKPFRFHPEHFLDAQGHFVKPEAFLPFSAGRRACLGEPLARMELFLFFTSLLQHFSFSVPTGQPRPSHHGVFAFLVTPSPYELCAVPR

TABLE 4 N-demethylase candidate enzymes SEQ ID Name Sequence NO: BM3MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAP 37GRVTRYLSSQRLIKEACDESRFDKNLSQAAKFARDFAGDGLVTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVSEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFIISMVRAADEVMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKARGEQSDDLLTQMLNGKDPETGEPLDDGNIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKVAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDEVMVLIPQLHRDKTVWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPKGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG CYP3A4-1MALIPDLAMETWLLLAVSLVLLYLYGTHSHGLFKKLGIPGPTPLPF 38LGNILSYHKGFCMFDMECHKKYGKVWGFYDGQQPVLAITDPDMIKTVLVKECYSVFTNRRPFGPVGFMKSAISIAEDEEWKRLRSLLSPTFTSGKLKEMVPIIAQYGDVLVRNLRREAETGKPVTLKDVFGAYSMDVITSTSFGVNIDSLNNPQDPFVENTKKLLRFDFLDPFFLSITVFPFLIPILEVLNICVFPREVTNFLRKSVKRMKESRLEDTQKHRVDFLQLMIDSQNSKETESHKALSDLELVAQSIIFIFAGYETTSSVLSFIMYELATHPDVQQKLQEEIDAVLPNKAPPTYDTVLQMEYLDMVVNETLRLFPIAMRLERVCKKDVEINGMFIPKGVVVMIPSYALHRDPKYWTEPEKFLPERFSKKNKDNIDPYIYTPFGSGPRNCIGMRFALMNMKLALIRVLQNFSFKPCKETQIPLKLSLGGLLQPEKPVVLKVESRDGTVSGA CYP3A4-2MALIPDLAMETWLLLAVSLVLLYLYGTHSHGLFKKLGIPGPTPLPF 39LGNILSYHKGFCMFDMECHKKYGKVWGFYDGQQPVLAITDPDMIKTVLVKECYSVFTNRRPFGPVGFMKSAISIAEDEEWKRLRSLLSPTFTSGKLKEMVPIIAQYGDVLVRNLRREAETGKPVTLKDVFGAYSMDVITSTSFGVNIDSLNNPQDPFVENTKKLLRFDFLDPFFLSIIFPFLIPILEVLNICVFPREVTNFLRKSVKRMKESRLEDTQKHRVDFLQLMIDSQNSKETESHKALSDLELVAQSIIFIFAGYETTSSVLSFIMYELATHPDVQQKLQEEIDAVLPNKAPPTYDTVLQMEYLDMVVNETLRLFPIAMRLERVCKKDVEINGMFIPKGVVVMIPSYALHRDPKYWTEPEKFLPERFSKKNKDNIDPYIYTPFGSGPRNCIGMRFALMNMKLALIRVLQNFSFKPCKETQIPLKLSLGGLLQPEKPVVLKVESRDGTVSGA McaCYP82-4MIMMFIDYYSSWLPQTLLLQSILLAVSLVIFINLFLTRRRSYSSKS 40HTNIIHPPKAAGALPVIGHLYTLFRGLSAGVPLYRQLDAMADRYGPAFIIHLGVYPTLVVTCRELAKECFTTNDQTFATRPSTCAGKYIGYNYAFFGFAPYGPYWREARKIATVELLSNYRLDSLRHVREAEVGRNVDELYALHASSSTNKQNMMKIDMKQWFDQVTLNVILMMVVGKRCVTTGGNEEEVRVVKVLHEFFKHLGTLSVSDVVPYVEWMDLDGNIGRMKSTAKELDCILGRWLEEHRRERRSDFMDAMLAMVEGIKIPYYDSDTVIKAICLNLLNAGSDTLGITMTWALSLLLNNRHVLKKVKDELDVHVGKNRQVEELDVKNLVYLHAVVKETLRLFPPAPLGVPHEAMEDCVVGGFHVAKGTRLVVNVWKLHRDPSVWSDPLAFKPERFLDNNTVDVRGQHFQLLPFGSGRRGCPGITFALQVAHLTLARLLHGFEWDTPDGAPVDMSEVSVLTTAKKNPVEVLFTPRLPAEVYTQN NsaCYP82-4MLSIHDSTMVFLQLQAICGIFGFIFIITWWTRWKSSNKMKAPEVAG 41AWPVIGHLHLLGGGRPLYQLLGDMSDKYGPAFTLRMGIQKALVVSSWEVAKECLTTNDRALATRPSSAGGKYMGYNNALIPFSPYGPYWRDMRKIATLELLSNHRLEELKHVREMEINTCISDMYKLCQVEDGVEIKPISVDLSQWFADLTFNVVVMMITGKRYIGSTDAGDMNEIRHFQAALVKFMRLLRISLLVDVFPVLQWINYGGFKGVMKSTARDIDSVLENWLQEHQRKRLSPDFNGNHDFIDVMISTLEGTEFSDYDHNTIIKAISMAMVVGGTDTTTTTLIWAISLLLNNPNAMKKVQEELEIHVGKERNVDGSDIQHLVYLQAVVKETLRLYPPVPLSVMHQAMEDCVIGSYNIQAGTRVLFNLWKLHRDSSVWSDPLEFRPERFLTSHVDVDVRGQHFELIPFGSGRRSCPGISFALQVIHLTIARLFHGFNLTTPGNSSVDMSEISGAT LSKVTPLEVLVTPRLSSKLYNHcaCYP82- MDSLLQLQIIGALAALIFTYKLLKVICRSPMTDGMEAPEPPGAWPI 42 10IGHLHLLGGQDPIARTLGVMTDKYGPILKLRLGVHTGLVVSNWELAKECFTTNDRVLASRPMGAAGKYLGYNYAIFGLAPHGPYWSEVRKIVLRELLSNQSLEKLKHVRISEINTCLKNLFSLNNGNTPIKVDMKQWFERPMFNVVTMMIAGKRYFSMENDNEAMNFRKVATEFMYLTGVFVVSDALPYLEWLDLQGHVSAMKRTAKELDIHVGKWLEEHRRAKLLGETKNEDDFVDVLLTILPEDLKDNQTYIHDRDTIIKATALALFLAASDTTAITLTWALSLILNNPDVLKRAQDELDKHVGKEKLVKESDIINLVYLQAIIKETLRLYPAAPLLLPHEAMEDCTVGGYHVPKGTRIFVNIWKLQRDPRVWFDPNEFRPERFLTTHANVDFKGQHFEYIPFSSGRRVCPGITFSTQIMHLTLAHLLHEFNIVTPTKSNAGVDMTESLGITMPKATPLEV LLTPRLPSNLYNQYRDEcaCYP82-7 MNLLIFFQFLLQFQVLVGLSVLLAFSYYLWVSKNPKINKFKGKGAL 43LAPQAAGAWPIVGHLPQLVGPKPLFRILGAMADNYGPIFMLRFGVHPTVVVSSWEMTKECFTTNDRHLASRPSNAASQYLIYEVYALFGFSLYGSSYWRDARKIATLELLSHRRLELLKHVPYTEIDTCIKQLHRLWTKNNKNQNNPELKVEMNQFFTDLTMNVILKLVVGKRFFNVDDAADHEKEEARKIQGTIFEFFKLTEGSVSAGALPLLNWLDLNGQKRAMKRTAKKMDSIAEKLLDEHRQKRLSKEGVKGTHDHNDFMDVLLSILDADQGDYSHHPFNYSRDHVIKATTLSMILSSMSISVSLSWALSLLLNNRHVLKKAQDELDMNVGKDRQVEEGDIKNLVYLQAIVKETFRMYPANPLLLPHEAIEDCKIGGFNVPAGTRVVVNAWKLQHDPRVWSNPSEFKPERFLNDQAAKVVDVRGQNFEYLPFGSGRRVCPGISFSLQTIHMSLARLVQAFELGTPSNERIDMTEGSGLTMPKTTPLHVLLNPRLPLPLYE GflCYP82-8MELINSLEIQPITISILALLTVSILLYKIIWNHGSRKNNKSNKNNRK 44TSSSAGVVEIPGAWPIIGHLHLFNGSEQMFHKLGSLADQYGPAPFFIRFGSRKYVVVSNWELVKTCFTAQSQIFVSRPPMLAMNILFFPKDSLSYIQHGDHWRELRKISSTKLLSSHRVETQKHLIASEVDYCFKQLYKLSNNGEFTLVRLNTWCEDMALNVHVRMIAGMKNYVAAPGSGEYGGQARRYRKALEEALDLLNQFTITDVVPWLGWLDHFRDVVGRMKRCGAELDSIFATWVEEHRVKRASGKGGDVEPDFIDLCWESMEQLPGNDPATVIKLMCKEHIFNGSGTSSLTLAWILSLIMNNPYVIKKAREELEKHVGNHRQVEESDLPNLLYIQAIIKEGMRLYTPGPFIDRNTTEDYEINGVHIPAGTCLYVNLWKIHRDPNVYEDPLEFKPERFLKNNSDLDLKGQNYQLLPFGAGRRICPGVSLALPLMYLTVSRLIHGFDMKLPKGVEKADMTAHGGVINQRAYPLEVLLKPRLTFQQA SdiCYP82-3MTIGALALLSFIYFLRVSVIKRTKYTNTAVTATNKLENDEDEANHSK 45RVVAPPEVAGAWPILGHLPQLVGLKQPLFRVLGDMADKYGPIFIVRFGMYPTLVVSSWEMAKECFTTNDRVLASRPASASGKYLTYNYAMFGFTNGPYWREIRKISMLELLSHRRVELLKHVPSTEIDSSIKQLYHLWVENQNQNKQGDHQVKVDMSQLLRDLTLNIVLKLVVGKRLFNNNDMDHEQDEAARKLQKTMVELIKVAGASVASDALPFLGWLDVDGLKRTMKRIAKEIDVIAERWLQEHRQKKLTSNDKGGSNNIQGGGGDNDFMDVMLSILDDDSNFFINYNRDTVIKATSLTMILAGSDTTTLSLTWALTLLATNPGALRKAQDELDTKVGRDRQVDERDIKNLVYLQAIVKETLRMYPAAPLAIPHEATQDCIVGGYHVTAGTRVWVNLWKLQRDPHAWPNPSEFRPERFLAVENDCKQQGTCDGEAANMDFRGQHFEYMPFGSGRRMCPGINFAIQIIHMTLARLLHSFELRVPEEEVIDMAEDSGLTI SKVTPLELLLTPRLPLPLYISdiCYP82-6 FCQFQGIVGILLAFLTFLYYLWRASITGLRTKPKHNDFKVTKAAPEA 46DGAWPIVGHFAQFIGPRPLFRILGDMADKYGSIFMVRFGMYPTLVVSSWEMAKECFTTNDRFLASRPASAAGKYLTYDFAMLSFSFYGPYWREIRKISMLELLSHRRVELLKHVPSTEIDSSIKQLYHLWVENQNQNKQGDHQVKVDMSQLLRDLTLNIVLKLVVGKRLFNNNDMDHEQDEAARKLQKTMVELIKVAGASVASDALPFLGWLDVDGLKRTMKRIAKEIDVIAERWLQEHRQKKLTSNDKGGSNNIQGGGGDNDFMDVMLSILDDDSNFFINYNRDTVIKATSLTMILAGSDTTTLSLTWALTLLATYPLCALRKAQDELDTKVGRDRQVDERDIKNLVYLQAIVKETLRMYPAAPLAIPHEATQDCIVGGYHVTAGTRVWVNLWKLQRDPHAWPNPSEFRPERFLAVENDCKQQGTCDGEAANMDFRGQHFEYMPFGSGRRMCPGINFAIQIIHMTLARLLHSFELRVPEEEVIDMAEDSGLTISKVTP LELLLTPRLPLPLYICmaCYP82-6 MDLFIFFSRFQYIVGLLAFLTFFYYLWRVSITGTRIKTNQNIMNGT 47NMMAPEAAGAWPIVGHLPQLVGPQPLFKILGDMADKYGSIFMVRFGMHPTLVVSSWEMAKECFTTNDKFLASRPTSAGGKYLTYDFAMFGFSFYGPYWREIRKISTLELLSHRRVELLKHVPYTEIGGSIKQLYKLWMETQNQNKQRDDHQVKVDMSQVFGYLTLNTVLKLVVGKGLFNNNDMNHEQEEGRKLHETVLEFFKLAGVSVASDALPFLGWLDVDGQKRSMKRIAKEMDLIAERWLQEHRQKRLTSNNKASSGHDDFMSVLLSILDDDSNFFNYNRDTVIKATSLNLILAASDTTSVSLTWVLSLLVTNPGALKKVQDELDTKVGRNRHVEERDIEKLVYLQATVKETLRMYPAGPLSVPHEATQDCTVGGYQVTAGTRLVVNVWKLQRDPRVWPNPSEFKPERFLPDGCEVGCGEAANMDFRGQHFEYIPFGSGRRMCPGIDFAIQIIHMTLACLLHAFEFQVPSSLDKHLVPAVIDMSEGSGLTMPKVTPLEV LLNPRLPLPLYEL EcaCYP82-5MEKPILLQLQPGILGLLALMCFLYYVIKVSLSTRNCNQLVRHPPEA 48AGSWPIVGHLPQLVGSGKPLFRVLGDMADKFGPIFMVRFGVHPTLVVSSWEMAKECFTSNDKFLASRPPSAASIYMAYDHAMLGFSSYGPYWREIRKISTLHLLSHRRLELLKHVPHLEIHNFIKGLYGIWKDHQKQQQQPTARDDQDSVMLEMSQLFGYLTLNIVLSLVVGKRVCNYHADGHLDDGEEAGQGQKLHQTITDFFKLSGVSVASDALPFLGLFDLDGQKKIMKRVAKEMDFVAERWLQDKKSSLLLSSKSNNKQNEAGEGDVDDFMDVLMSTLPDDDDSFFTKYSRDTVIKANSLSMVVAGSDTTSVSLTWALSLLLNNIQVLRKAQDELDTKVGRDRHVEEKDIDNLVYLQAIVKETLRMYPAGPLSVPHEAIEDCNVGGYHIKTGTRLLVNIWKLQRDPRVWSNPSEFRPERFLDNQSNGTLLDFRGQHFEYIPFGSGRRMCPGVNLATPILHMTLARLLQSFDLTTPSSSPVDMTEGSGLTMPKVTPLKV LLTPRLPLPLYDY PbrCYP82-5MDVAIIVDHHYLQPFVSIAGLLALLSFFYCIWVFIIRPRIIKSNLD 49ERKLSPSSPPEVAGAWPIVGHLPQLIGSTPLFKILADMSNKYGPIFMVRFGMYPTLVVSSWEMSKECFTTNDRLFATRPPSAAGKYLTKALFAFSVYGPYWREIRKISTIHLLSLRRLELLKHGRYLEIDKCMKRLFEYWMEHHKNIISTTSSVKVNMSQVFAELSLNVVLKIIVGKTLFIKNGNEDYTKEEEEGQKLHKTILKFMELAGVSVASDVLPFLGWLDVDGQKKQMKRVYKEMNLIASKWLGEHRERKRLQIIQKRGAARGSNYDDGNDFMDVLMSILDEENDDLFFGYSRDTVIKSTCLQLIVAASDTTSLAMTWALSLLLTNPNVLQKAQDELDTKVGRDRIIEEHDIECLVYLQAIVKETLRLYPPAPLSLPHEAMEDCTVGGYQVKAGTRLVVNLWKLQRDPRVWSNPLEFKPERFLPQSDGGFGGEEARMDFRGQHFEYTPFGSGRRICPGIDFFLQTVHMALARLLQAFDFNTAGGLVIDMVEGPGLTMPKVT PLEVHLNPRLPVTLYPbrCYP82-6 MQVDWPNILQKYYPIITCSLLTLLSFYYIWVSITKPSRNSKTKLPP 50PEVAGSWPIVGHLPQLVGSTPLFKILANMSDKYGPIFMVRFGMHPTLVVSSWEMSKECFTTNDKFLASRPPSASAKYLGYDNAMFVFSDYGPYWREIRKISTLQLLTHKRLDSLKNIPYLEINSCVKTLYTRWAKTQSQIKQNVGGAADDFVKVDMTEMFGHLNLNVVLRLVVGKPIFIQKDNADEDYTKDGHNKEELGQKLHKTIIEFFELAGASVASDVLPYLGWLDVDGQKKRMKKIAMEMDLFAQKWLEEHRQKGINHDNENDFMAVLISVLGEGKDDHIFGYSRDTVIKATCLTLIVAATDTTLVSLTWALSLLLTNPRVLSKAQDELDTVVGKERNVEDRDVNHLVYLQAVIKETLRLYPPSPLAVPHEAIENCNVGGYEVKARTRLLVNLWKIHRDPRVWSNPLEFKPERFLPKLDGGTGEASKLDFKGQDFVYTPFGSGRRMCPGINFASQTLHMTLARLLHAFDFDIESNGLVIDMTEGSGLTMPKVTPLQVHLRPR LPATLY McaCYP82-4MIMMFIDYYSSWLPQTLLLQSILLAVSLVIFINLFLTRRRSYSSKS 51HTNIIHPPKAAGALPVIGHLYTLFRGLSAGVPLYRQLDAMADRYGPAFIIHLGVYPTLVVTCRELAKECFTTNDQTFATRPSTCAGKYIGYNYAFFGFAPYGPYWREARKIATVELLSNYRLDSLRHVREAEVGRNVDELYALHASSSTNKQNMMKIDMKQWFDQVTLNVILMMVVGKRCVTTGGNEEEVRVVKVLHEFFKHLGTLSVSDVVPYVEWMDLDGNIGRMKSTAKELDCILGRWLEEHRRERRSDFMDAMLAMVEGIKIPYYDSDTVIKAICLNLLNAGSDTLGITMTWALSLLLNNRHVLKKVKDELDVHVGKNRQVEELDVKNLVYLHAVVKETLRLFPPAPLGVPHEAMEDCVVGGFHVAKGTRLVVNVWKLHRDPSVWSDPLAFKPERFLDNNTVDVRGQHFQLLPFGSGRRGCPGITFALQVAHLTLARLLHGFEWDTPDGAPVDMSEVSVLTTAKKNPVEVLFTPRLPAEVYTQN NsaCYP82-4MLSIHDSTMVFLQLQAICGIFGFIFIITWWTRWKSSNKMKAPEVAG 52AWPVIGHLHLLGGGRPLYQLLGDMSDKYGPAFTLRMGIQKALVVSSWEVAKECLTTNDRALATRPSSAGGKYMGYNNALIPFSPYGPYWRDMRKIATLELLSNHRLEELKHVREMEINTCISDMYKLCQVEDGVEIKPISVDLSQWFADLTFNVVVMMITGKRYIGSTDAGDMNEIRHFQAALVKFMRLLRISLLVDVFPVLQWINYGGFKGVMKSTARDIDSVLENWLQEHQRKRLSPDFNGNHDFIDVMISTLEGTEFSDYDHNTIIKAISMAMVVGGTDTTTTTLIWAISLLLNNPNAMKKVQEELEIHVGKERNVDGSDIQHLVYLQAVVKETLRLYPPVPLSVMHQAMEDCVIGSYNIQAGTRVLFNLWKLHRDSSVWSDPLEFRPERFLTSHVDVDVRGQHFELIPFGSGRRSCPGISFALQVIHLTIARLFHGFNLTTPGNSSVDMSEISGAT LSKVTPLEVLVTPRLSSKLYNHcaCYP82- MDSLLQLQIIGALAALIFTYKLLKVICRSPMTDGMEAPEPPGAWPI 53 10IGHLHLLGGQDPIARTLGVMTDKYGPILKLRLGVHTGLVVSNWELAKECFTTNDRVLASRPMGAAGKYLGYNYAIFGLAPHGPYWSEVRKIVLRELLSNQSLEKLKHVRISEINTCLKNLFSLNNGNTPIKVDMKQWFERPMFNVVTMMIAGKRYFSMENDNEAMNFRKVATEFMYLTGVFVVSDALPYLEWLDLQGHVSAMKRTAKELDIHVGKWLEEHRRAKLLGETKNEDDFVDVLLTILPEDLKDNQTYIHDRDTIIKATALALFLAASDTTAITLTWALSLILNNPDVLKRAQDELDKHVGKEKLVKESDIINLVYLQAIIKETLRLYPAAPLLLPHEAMEDCTVGGYHVPKGTRIFVNIWKLQRDPRVWFDPNEFRPERFLTTHANVDFKGQHFEYIPFSSGRRVCPGITFSTQIMHLTLAHLLHEFNIVTPTKSNAGVDMTESLGITMPKAT PLEVLLTPRLPSNLYNQYRDEcaCYP82-7 MNLLIFFQFLLQFQVLVGLSVLLAFSYYLWVSKNPKINKFKGKGAL 54LAPQAAGAWPIVGHLPQLVGPKPLFRILGAMADNYGPIFMLRFGVHPTVVVSSWEMTKECFTTNDRHLASRPSNAASQYLIYEVYALFGFSLYGSSYWRDARKIATLELLSHRRLELLKHVPYTEIDTCIKQLHRLWTKNNKNQNNPELKVEMNQFFTDLTMNVILKLVVGKRFFNVDDAADHEKEEARKIQGTIFEFFKLTEGSVSAGALPLLNWLDLNGQKRAMKRTAKKMDSIAEKLLDEHRQKRLSKEGVKGTHDHNDFMDVLLSILDADQGDYSHHPFNYSRDHVIKATTLSMILSSMSISVSLSWALSLLLNNRHVLKKAQDELDMNVGKDRQVEEGDIKNLVYLQAIVKETFRMYPANPLLLPHEAIEDCKIGGFNVPAGTRVVVNAWKLQHDPRVWSNPSEFKPERFLNDQAAKVVDVRGQNFEYLPFGSGRRVCPGISFSLQTIHMSLARLVQAFELGTPSNERIDMTEGSGLTMPKTTPLHVLLNPRLPLPL YE GflCYP82-8MELINSLEIQPITISILALLTVSILLYKIIWNHGSRKNNKSNKNNR 55KTSSSAGVVEIPGAWPIIGHLHLFNGSEQMFHKLGSLADQYGPAPFFIRFGSRKYVVVSNWELVKTCFTAQSQIFVSRPPMLAMNILFFPKDSLSYIQHGDHWRELRKISSTKLLSSHRVETQKHLIASEVDYCFKQLYKLSNNGEFTLVRLNTWCEDMALNVHVRMIAGMKNYVAAPGSGEYGGQARRYRKALEEALDLLNQFTITDVVPWLGWLDHFRDVVGRMKRCGAELDSIFATWVEEHRVKRASGKGGDVEPDFIDLCWESMEQLPGNDPATVIKLMCKEHIFNGSGTSSLTLAWILSLIMNNPYVIKKAREELEKHVGNHRQVEESDLPNLLYIQAIIKEGMRLYTPGPFIDRNTTEDYEINGVHIPAGTCLYVNLWKIHRDPNVYEDPLEFKPERFLKNNSDLDLKGQNYQLLPFGAGRRICPGVSLALPLMYLTVSRLIHGFDMKLPKGVEKADMTAHGGVINQRAYPLEVLLKPRLTFQQA SdiCYP82-3MTIGALALLSFIYFLRVSVIKRTKYTNTAVTATNKLENDEDEANHS 56KRVVAPPEVAGAWPILGHLPQLVGLKQPLFRVLGDMADKYGPIFIVRFGMYPTLVVSSWEMAKECFTTNDRVLASRPASASGKYLTYNYAMFGFTNGPYWREIRKISMLELLSHRRVELLKHVPSTEIDSSIKQLYHLWVENQNQNKQGDHQVKVDMSQLLRDLTLNIVLKLVVGKRLFNNNDMDHEQDEAARKLQKTMVELIKVAGASVASDALPFLGWLDVDGLKRTMKRIAKEIDVIAERWLQEHRQKKLTSNDKGGSNNIQGGGGDNDFMDVMLSILDDDSNFFINYNRDTVIKATSLTMILAGSDTTTLSLTWALTLLATNPGALRKAQDELDTKVGRDRQVDERDIKNLVYLQAIVKETLRMYPAAPLAIPHEATQDCIVGGYHVTAGTRVWVNLWKLQRDPHAWPNPSEFRPERFLAVENDCKQQGTCDGEAANMDFRGQHFEYMPFGSGRRMCPGINFAIQIIHMTLARLLHSFELRVPEEEVIDMAEDSGL TISKVTPLELLLTPRLPLPLYISdiCYP82-6 FCQFQGIVGILLAFLTFLYYLWRASITGLRTKPKHNDFKVTKAAPE 57ADGAWPIVGHFAQFIGPRPLFRILGDMADKYGSIFMVRFGMYPTLVVSSWEMAKECFTTNDRFLASRPASAAGKYLTYDFAMLSFSFYGPYWREIRKISMLELLSHRRVELLKHVPSTEIDSSIKQLYHLWVENQNQNKQGDHQVKVDMSQLLRDLTLNIVLKLVVGKRLFNNNDMDHEQDEAARKLQKTMVELIKVAGASVASDALPFLGWLDVDGLKRTMKRIAKEIDVIAERWLQEHRQKKLTSNDKGGSNNIQGGGGDNDFMDVMLSILDDDSNFFINYNRDTVIKATSLTMILAGSDTTTLSLTWALTLLATYPLCALRKAQDELDTKVGRDRQVDERDIKNLVYLQAIVKETLRMYPAAPLAIPHEATQDCIVGGYHVTAGTRVWVNLWKLQRDPHAWPNPSEFRPERFLAVENDCKQQGTCDGEAANMDFRGQHFEYMPFGSGRRMCPGINFAIQIIHMTLARLLHSFELRVPEEEVIDMAEDSGLTISKVTP LELLLTPRLPLPLYICmaCYP82-6 MDLFIFFSRFQYIVGLLAFLTFFYYLWRVSITGTRIKTNQNIMNGT 58NMMAPEAAGAWPIVGHLPQLVGPQPLFKILGDMADKYGSIFMVRFGMHPTLVVSSWEMAKECFTTNDKFLASRPTSAGGKYLTYDFAMFGFSFYGPYWREIRKISTLELLSHRRVELLKHVPYTEIGGSIKQLYKLWMETQNQNKQRDDHQVKVDMSQVFGYLTLNTVLKLVVGKGLFNNNDMNHEQEEGRKLHETVLEFFKLAGVSVASDALPFLGWLDVDGQKRSMKRIAKEMDLIAERWLQEHRQKRLTSNNKASSGHDDFMSVLLSILDDDSNFFNYNRDTVIKATSLNLILAASDTTSVSLTWVLSLLVTNPGALKKVQDELDTKVGRNRHVEERDIEKLVYLQATVKETLRMYPAGPLSVPHEATQDCTVGGYQVTAGTRLVVNVWKLQRDPRVWPNPSEFKPERFLPDGCEVGCGEAANMDFRGQHFEYIPFGSGRRMCPGIDFAIQIIHMTLACLLHAFEFQVPSSLDKHLVPAVIDMSEGSGLTMPKVTPLEV LLNPRLPLPLYEL EcaCYP82-5MEKPILLQLQPGILGLLALMCFLYYVIKVSLSTRNCNQLVRHPPEA 59AGSWPIVGHLPQLVGSGKPLFRVLGDMADKFGPIFMVRFGVHPTLVVSSWEMAKECFTSNDKFLASRPPSAASIYMAYDHAMLGFSSYGPYWREIRKISTLHLLSHRRLELLKHVPHLEIHNFIKGLYGIWKDHQKQQQQPTARDDQDSVMLEMSQLFGYLTLNIVLSLVVGKRVCNYHADGHLDDGEEAGQGQKLHQTITDFFKLSGVSVASDALPFLGLFDLDGQKKIMKRVAKEMDFVAERWLQDKKSSLLLSSKSNNKQNEAGEGDVDDFMDVLMSTLPDDDDSFFTKYSRDTVIKANSLSMVVAGSDTTSVSLTWALSLLLNNIQVLRKAQDELDTKVGRDRHVEEKDIDNLVYLQAIVKETLRMYPAGPLSVPHEAIEDCNVGGYHIKTGTRLLVNIWKLQRDPRVWSNPSEFRPERFLDNQSNGTLLDFRGQHFEYIPFGSGRRMCPGVNLATPILHMTLARLLQSFDLTTPSSSPVDMTEGSGLTMPKVTPLKV LLTPRLPLPLYDY PbrCYP82-5MDVAIIVDHHYLQPFVSIAGLLALLSFFYCIWVFIIRPRIIKSNLD 60ERKLSPSSPPEVAGAWPIVGHLPQLIGSTPLFKILADMSNKYGPIFMVRFGMYPTLVVSSWEMSKECFTTNDRLFATRPPSAAGKYLTKALFAFSVYGPYWREIRKISTIHLLSLRRLELLKHGRYLEIDKCMKRLFEYWMEHHKNIISTTSSVKVNMSQVFAELSLNVVLKIIVGKTLFIKNGNEDYTKEEEEGQKLHKTILKFMELAGVSVASDVLPFLGWLDVDGQKKQMKRVYKEMNLIASKWLGEHRERKRLQIIQKRGAARGSNYDDGNDFMDVLMSILDEENDDLFFGYSRDTVIKSTCLQLIVAASDTTSLAMTWALSLLLTNPNVLQKAQDELDTKVGRDRIIEEHDIECLVYLQAIVKETLRLYPPAPLSLPHEAMEDCTVGGYQVKAGTRLVVNLWKLQRDPRVWSNPLEFKPERFLPQSDGGFGGEEARMDFRGQHFEYTPFGSGRRICPGIDFFLQTVHMALARLLQAFDFNTAGGLVIDMVEGPGLTMPKVT PLEVHLNPRLPVTLYPbrCYP82-6 MQVDWPNILQKYYPIITCSLLTLLSFYYIWVSITKPSRNSKTKLPP 61PEVAGSWPIVGHLPQLVGSTPLFKILANMSDKYGPIFMVRFGMHPTLVVSSWEMSKECFTTNDKFLASRPPSASAKYLGYDNAMFVFSDYGPYWREIRKISTLQLLTHKRLDSLKNIPYLEINSCVKTLYTRWAKTQSQIKQNVGGAADDFVKVDMTEMFGHLNLNVVLRLVVGKPIFIQKDNADEDYTKDGHNKEELGQKLHKTIIEFFELAGASVASDVLPYLGWLDVDGQKKRMKKIAMEMDLFAQKWLEEHRQKGINHDNENDFMAVLISVLGEGKDDHIFGYSRDTVIKATCLTLIVAATDTTLVSLTWALSLLLTNPRVLSKAQDELDTVVGKERNVEDRDVNHLVYLQAVIKETLRLYPPSPLAVPHEAIENCNVGGYEVKARTRLLVNLWKIHRDPRVWSNPLEFKPERFLPKLDGGTGEASKLDFKGQDFVYTPFGSGRRMCPGINFASQTLHMTLARLLHAFDFDIESNGLVIDMTEGSGLTMPKVTPLQVHLRPR LPATLY PbrCYP82-7MMDLAMFIDQYFSLAKIAGLLALLSFFYYLWISTLWSPRNPKLSSV 62SPPEVAGAWPILGHLPQLLGSRPLFKILADMSDNYGPIFMVRFGMHPTLVVSSWEMAKECFTTNDRFLAGRPSGAANKYLTFALFGFSTYGPYWREIRKIATLHLLSHRRLELLKHVPDLEVTNCMKHLHRRWIDSQNQIKQNDAAAGSVKVDMGRVFGELTLNVVLKLVAGKSIFFKNDNTRQYDSKDGHNKEEEEGKKLHKTIIDFYSLAGASVASDVLPFLGWLDVDGQKKRMKRVAKDMDFIAAKWLEEHRHQKRQTVLSSSATLGSSNHDDAKDFMDVLMSILDGENDDLFFGYSRDTVIKTTCLQLIAAAADTTSVTMTWALALLITNPTILRKAQDELDTKVGKDRNIEERDINDLVYLQAIVKETLRMYPAGPLNVPHEAIADCNIGGYEVRAGTRLLVNLWKMHRDPRVWSNPSEFKPERFLPQLDGGSGGEAANLDFRGQDFEYLPFSAGRRMCPGIDFSLQTLHMTLARLLHGFDFNNDSAGIIIDMEEGSG LTMPKLTPLEIYLCPRLPAKLY

TABLE 5 N-methyltransferase and N-modifying candidate enzymes SEQ IDName Sequence NO: TfCNMT MAVEGKQVAPKKAIIVELLKKLELGLVPDDEIKKLIRIQLGRRLQWG63 CKSTYEEQIAQLVNLTHSLRQMKIATEVETLDDQMYEVPIDFLKIMNGSNLKGSCCYFKNDSTTLDEAEIAMLELYCERAQIKDGHSVLDLGCGQGALTLYVAQKYKNSRVTAVTNSVSQKEFIEEESRKRNLSNVEVLLADITTHKMPDTYDRILVVELFEHMKNYELLLRKIKEWMAKDGLLFVEHICHKTFAYHYEPIDEDDWFTEYVFPAGTMIIPSASFFLYFQDDVSVVNHWTLSGKHFSRTNEEWLKRLDANVELIKPMFVTITGQCRQEAMKLINYWRGFCLSGMEMFGYNNGEEWMASHVLFKKK CjCNMTMAVEAKQTKKAAIVELLKQLELGLVPYDDIKQLIRRELARRLQWGY 64KPTYEEQIAEIQNLTHSLRQMKIATEVETLDSQLYEIPIEFLKIMNGSNLKGSCCYFKEDSTTLDEAEIAMLDLYCERAQIQDGQSVLDLGCGQGALTLHVAQKYKNCRVTAVTNSVSQKEYIEEESRRRNLLNVEVKLADITTHEMAETYDRILVIELFEHMKNYELLLRKISEWISKDGLLFLEHICHKTFAYHYEPLDDDDWFTEYVFPAGTMIIPSASFFLYFQDDVSVVNHWTLSGKHFSRTNEEWLKRLDANLDVIKPMFETLMGNEEEAVKLINYWRGFCLSGMEMFGYNNGEEWMASHVLFKKK PsCNMTMQLKAKEELLRNMELGLIPDQEIRQLIRVELEKRLQWGYKETHEEQL 65SQLLDLVHSLKGMKMATEMENLDLKLYEAPMEFLKIQHGSNMKQSAGYYTDESTTLDEAEIAMLDLYMERAQIKDGQSVLDLGCGLGAVALFGANKFKKCQFTGVTSSVEQKDYIEGKCKELKLTNVKVLLADITTYETEERFDRIFAVELIEHMKNYQLLLKKISEWMKDDGLLFVEHVCHKTLAYHYEPVDAEDWYTNYIFPAGTLTLSSASMLLYFQDDVSVVNQWTLSGKHYSRSHEEWLKNMDKNIVEFKEIMRSITKTEKEAIKLLNFWRIFCMCGAELFGYKNGEEWMLTHLLFKKK PsTNMTMGSIDEVKKESAGETLGRLLKGEIKDEELKKLIKFQFEKRLQWGYKS 66SHQEQLSFNLDFIKSLKKMEMSGEIETMNKETYELPSEFLEAVFGKTVKQSMCYFTHESATIDEAEEAAHELYCERAQIKDGQTVLDIGCGQGGLVLYIAQKYKNCHVTGLTNSKAQVNYLLKQAEKLGLTNVDAILADVTQYESDKTYDRLLMIEAIEHMKNLQLFMKKLSTWMTKESLLFVDHVCHKTFAHFFEAVDEDDWYSGFIFPPGCATILAANSLLYFQDDVSVVDHWVVNGMHMARSVDIWRKALDKNMEAAKEILLPGLGGSHETVNGVVTHIRTFCMGGYEQFSMNNGDEWMVAQLLFKKK EcTNMTMGSSAGEIMGRLMKGEIEDEELKKLIRHQWDRRIEWGYKPTHEKQL 67AFNLDFIKGLKEMVMSGEIDTMNKETYELPTAFLEAVFGKTVKQSCCYFKDENSTIDEAEEAAHELYCERAQIKDGQTVLDIGCGQGGLVLYIAEKYKNCHVTGLTNSKAQANYIEQQAEKLELTNVDVIFADVTKFDTDKTYDRILVVETIEHMKNIQLFMKKLSTWMTEDSLLFVDHISHKTFNHNFEALDEDDWYSGFIFPKGCVTILSSSTLLYFQDDVSALDHWVVNGMHMARSVEAWRKKLDETIEAAREILEPGLGSKEAVNQVITHIRTFCI GGYEQFSYNNGEEWMITQILFKKKPsRNMT MSTTMETTKISQQDDLWKNMELGQISDEEVRRLMKIGIEKRIKWGTK 68PTQQEQLAQLLDFNKSLRGMKMATEIDTLENHKIYETPESFNQIIGGKESAGLFTDETTTTMEEANTKMMDLYCERAGLKDGHTILDLGCGAGLLVLHLAKKYKKSKITGITNTSSHKEYILKQCKNLNLSNVEIILADVTKVDIESTFDRVFVIGLIEHMKNFELFLRKISKWMKDDGLLLLEHLCHKSFSDHWEPLSEDDWYAKNFFPSGTLVIPSATCLLYFQEDVTVTDHWILSGNNFARSNEVILKRIDGKIEEVKDIFMSFYGIGREEAVKLINWWRLLCITANELFKYNNGEEWLISQLLFKKKLMTCI TfPNMTMETKQTKKEAVANLIKRIEHGEVSDEEIRGMMKIQVQKRLKWGYKP 69THEQQLAQLVTFAQSLKGMEMAEEVDTLDAELYEIPLPFLHIMCGKTLKFSPGYFKDESTTLDESEVYMMDLYCERAQIKDGQSILDLGCGHGSLTLHVAQKYRGCKVTGITNSVSQKEFIMDQCKKLDLSNVEIILEDVTKFETEITYDRIFAVALIEHMKNYELFLKKVSTWIAQYGLLFVEHHCHKVFAYQYEPLDEDDWYTEYIFPSGTLVMSSSSILLYFQEDVSVVNHWTLSGKHPSLGFKQWLKRLDDNIDEVKEIFESFYGSKEKAMKFITYWRVFCIAHSQMYSTNNGEEWMLSQVLFKKK PbrTNMT1MGSIDEVKKESAGETLGRLLKGEIKDEELKKLIKFQFEKRLQWGYKS 70SHQEQLSFNLDFIKSLKKMEMSGEIETMNKETYELPSEFLEAVFGKTVKQSMCYFKHESATIDEAEEAAHELYCERAQIKDGQTVLDIGCGQGGLVLYIARKYKKCHVTGLTNSKAQVNYLLKQAEKLGLTNVDAILADVTQYESDKTYDRLLMIEAIEHMKNLQLFMKKLSTWMTEESLLFVDHVCHKTFAHFFEAVDEDDWYSGFIFPPGCATILAANSLLYFQDDVSVVDHWVVNGMHMARSVDIWRKALDKNMEAAKEILLPGLGGSHEAVNGVVTHIRTFCMGGYEQFSMNDGDEWMVAQLLFKKK PbrTNMT2MGSIEEVKKESAEETLGRLLRGEINDEELKKLIKYQLEKRLQWGYKS 71SHQEQLSFNLDFINSLKKMGMSGQVEAFTNEVYELPTECFEAAYGKSMKLSGCYFKHESSTIDEAEEASHELYCERAQIKDGQTVLDIGCGQGGLVLYVAQKYKNCHVTGLTNSKEQVNYILKQAEKLGLRNVDVILADVTQYESDKTYDRILVIGVVEHMKNMQLFIKKLSTWMAEDSLLFVDHSCHKTFNHFFEALDEDDWYSGYIFPPGCATFLSADSLLYFQDDVSVVDHWVVNGMHFARTVDAWRKKLDKNMEAVKEILLPGLGGNHEAVNGVITHIRTCCVGGYVQFSLNDGDEWMNAQLLFKKK AmcNMT1MCLFFAEKMGLMAEANNQQQLKKEDLLKNMELGLIPDEEIRKLIRV 72QLEKRLNWGYKSTHEQQLSQLLHLVHSLKKMKIATEMENLDLKLYEAPFSFVQIQHGSTIKESSGLFKDESTTLDEAEIAMLDLYTKRAKIEDGQSVLDLGCGLGAVTLYVAQKFKNCYVTGITSSVEQKDFIEGRCKELKLSNVKVILADITTYETEEKYNRIFAVELIEHMKNYELLLRKISEWMKQDGLLFIEHVCHKTLAYHYEPLDEEDWYTNYIFPAGTLTLSSATLLLYFQDDVAVVDQWTLSGKHYSRSHEEWLKRIDGNIEEVKEIMKSITKSEEEAKKLLNFWRIFCMCGAELFGYKNGEEWMMTHILFKKK GflNMT1MDLMATSKQVKKKEELLKNMELGLVPDEEIRRLIRIELEKRLKWGY 73KPTHQQQLAQLLDLVHSLKKMKIATEMESLDLKLYEAPFSFVQIKHGSTIKESSSYFKDESMTLDEAEIAMLDLYVERAQIEDGQSVLDLGCGLGAVTLHVAKKYKNCHVTGLTNSVEQKDFIEGKCKELNLSNVKVILADVTSHEMEDKFDRIFAVELIEHMKNYELLLRRISKWMKDDGLLFIEHVCHKTFAYHYEPIDEDDWYTEYIFPAGTLTLSSASLLLYFQDDVSVVNHWTLSGKHYSRSHEEWLKRIDGNMDAVKEIMKSITKTEEEAVKLINFWRIFCMCGAELFGYKDGEEWMMSHVLFKKKQLLQQC EcaNMT1MVDLKVEKEELLKSMELGLVPDEDIRKHIRSQLEKRLKWGYKPNHE 74QQLAQLLDVIHSLKKMKISKEYESFDLRLYEAPFDFHKIQLGTHLKESCSYYKDESTTLDEAEGAMLDLYTQKAKIEDGQSILDLGCGVGAVTLFVANKYKNCKVTGITSCQWQKDFIENKCKELNLTNVRVIIGDVTAYEMEETFDRIFAIELIEHMKNYELLLRKISKWMKDDGLLFIEHVCHKILAYPYEPIDEEDWFTEYIFPGGTLTLSSASLLLYFQDDVSVVEHSSLNGKHYSRSHGEWLKNIDANIDEVKGIMRSITKTEEEAVRLVNFWRIFCMC GIELFGYNNGEEWMVSHILLKKKEcaNMT2 MAADLVVKKWNNKKELIDEMELGLVGDEEIRELIRNDLEKRLKWG 75YKSNHEQQLAQLLHFVHSLRGMKIAADEVESFNIKVYEAPFSFNKIQLGSSLKESSCYYKHDETTLDEGEIAMMELYTEKAQIKDGQSVLDLGCGLGSLTLYVANKYPNCKVTGTTASLWHKDFIESKCKEQELTNVKIVLGDATTHEMEERFDRILAIGLIEHLKNYGLLLGRISKWLKDDGFLFIQHVCHKTLAYPLVPVDEEDWIGEYIFPGGTLTMPSASLLLYFQDELSVVDHSTLNGKHFSRTHEEWLKNIDAKIDEVKEILKSVTKTEEEVVRLTNFWRIFCMFGVEMFGYNEGEEWMLSQILFKKK CmaNMT4MASGKVVDLLKRLDSGLVSDEELRRVIRFELERRLKWGYKPTHEQQL 76AELLNLAHATKQMEIATKIDTLNSTMYEVPNSFLEIQLGSTLKESCLYFKDESTTVDEAEIAMMDLYLERAQIKDGQIILDLGCGLGALAFHIAQKYTNCNVTSVTNSVKQKEFIEEKCKILNVSNVKVILTDICTLEMEATFDRIFAIGLIEHMKNYELLLRKFSAWMKQDGLLFIEHLCHKTLGYHNEPIDEDDWYTAYFFPAGTLTFIPSSFLLYFQDDVSVVNHWTLSGKHFSRSNEEWLKRMDNKIDEVKEIYKAAASETKDDDIMKLIRLWRFLSISA AEMFGYKDGEEWMISQVLFKKKEcNMT3 MASLVEEGSFVNNKESVKERVSELVKRLKNGLVSDEELRKLMRVEL 77EKRLEWGYKSTHEQQLSQLIDLAHSMKKMEIAMEIDALNSTVYEVPLSFLQIIHGTTIKESCLYFKDESTTVDEAEIAMMDLYLERAQIKDGQSILDLGCGLGGFSFHIASKFTGCNITAVTNSVKQKEFIEEKCKTLNVPNIKVILADICTTEIENVFDRIIAIGLIEHMKNYELLLKKFSKWMTQDGLLFIEHLCHKTFGYHNEPLDEDDWYTTYFFPAGTLTFIPSSFLLYFQDDVSVVDHWTLNGKHFARSNEEWLKRMDEKMDEVKQIFRSNLKSENEVTKTIGEWRFLSMSAAEMFGYNNGEEWMVSQLLFKKK GflNMT5MGSNETNGELKTKEMVPDLLKRLESGLVADEELRKLIRFELERRLK 78WGYKPTHEQQLAELLKLAHSTKQMKIATETDSLNSTMYEVPIPFLQLQFGSAIKESCCYFKDESTTLDEAEVAMMDLYLERTQIKDGQSILDLGCGLGALAFHIVQKYPNCNVLAITNSVEQKEFIEEKCKIRKVENVKVSLADICTLEMKTTFDRIFAIGLLEHMKNYQLLLKKFSNWMKQDGLLFIEHLCHKTLAYHYEPLDEDDWYTEYFFPAGTLTIISSSFLLYFQDDVSIVNHWSLSGKHFSRSNEEWLKRMDMKIDEVKEILEAAFENKDHDITKLtNHWRFLAlNATEMFGYNNGEEWMVSQVLFKKK ScaNMT1MASDHEVSNKELKKKKEVITELLKRLESGLVSDEELRGLIRFELERRL 79RWGYKPTHEQQLAQLLNLAHSMKQMKIATEIDALNSTMYEVPIPFLQIQLGSTLKESCCYFKDESTTVDEAEIAMMDLYLERAQIKDGQSILDLGCGLGALAFHIAQKYTNCNITAITNSVRQKEFIEEKCKILNVSNVKVSLADICTLEMEATFDRIFAIGLIEHMKNYELLLKKFSEWMKQDGLIFIEHLCHKTLAYHYEPLDEDDWYTEYFFPAGTLTLISSSFLLYFQDDVSVVDHWTLSGKHFSRSNEEWLKRMDEKIDEVKEIFESVSDSKDDDVTKLINHWRFFCISSAEMFGYNNGEEWMISQVLFKKK CchNMT3MIKKSKIMAFSDHHHEVVKNHSKKEMIADLLKRLEAGLVPDEEMRN 80LFRFELERRLQWGYKSIHQEQLSQLLKLAHSTKEMTIVAEMDALNSSMYELPISFLQIQLGSNLKQSSLYFKDELTTVDEAEVAIMDLYLERAQIEDGQSILDLGCGLGAFSFHVARKYTNCNITAVTNSLTQKEFIEKKSKILNIQNVKVIFADVTTVEMETTFDRVFAIGLIEHMQNYELFLKKLSKWMKQDGLLFIEHFCHKTLAYHYKPIDEDDWFTNLLYPNGTVISSSLLLYFQDDVSVVDHWSLSGKHFSRASEESLKRMDAKMDEMKEIFESITDSKEEAMKLINQWRIFCISCAEMFGYNNGEEWMTSHFLFKKKL CchNMT6MGSSTASDHEMVIMENDSKNKQVVIADLLKRLVGGLVPDEEMRNM 81FRFELEKRLKWGYKSTHQQQLSQLLNLVELNKGIAKIAPEMDALNSAMYEVPIPYLKLMLGSTLKQSCLYFKDESTTLDEAEIEMMDLYLERADIQDGQSILDLGCGLGGLGFHIAQKYISCNITALTNSLTQKEFIEEKCKTLNIPNVKVILADVTTVEIETTFDRLFAIGLVEHMENYELFLRKLSKWMKQDGLLFIEHLCHKTLAYHYKPIDEDDWYSNLLYPTGTLTSASFLLYFQDDLSVVDHWSLSGKHFSRATEEWLKMIDANMDKIREIYESVTESKEEATRSINQWRIFCISCAEMFGYNDGEEWMISHFLFKNKKQIE CchNMT1MATSDQEVKTSKMEMIADLLKRLEAGLVPDDEIRSLIRVELERRLKW 82GYKSTHQEQLDQLLNLAHSIKKMKIASTEMDGLTSTMYEVPISLVQIQLGSHLKESCLYFKDETTTVDEAEIAMMDLYLERAQIKDGQSILDLGCGLGAVSFHIAQKYTSCNITAVTNSVRQKEFIEEKSKTLNVPNVKVLLADITTLEMEHTFDRLFAISLIEHMENYELLLRKLSEWMKQDGLLFIEHLCHKTLSYHFEPMDEDDWYTNLLFPAGTLTLVSASFLLYFQDDLSVVNQWVMSGKHFSRANEEWLKNMDAKMDEMREIFESITDSEEEVVKLINHWRIFCISSAEMFAYNDGEEWMNSHVLFKKKKQIQ CchNMT2MAGSGANKEMIADLLKRLEVGLVPDEEIRSLIRFQLKRRLKWGYKTT 83HQEQLEQLLSLAHSIRKMKIATEMDALNSTMYEVPISFMQIVFGSTLKESCLYFKDEATTVNEAEIAMMDLYLERAQIKDGQSILDLGCGMGSLCFHIARKYTNCNITAVTNSVSQKEFIEEKSKTLNLPNVKVILADITTLEMDDTYDCLFAIGLIEHMKNYELLLRKLSNWMKQDSLLFIDHVCHKTLAYHYEPIDEDDWYTNLLFPAGTLTLVSASFLLYFQDDLSLVDHWSMSGKHFSRTNKEWLKNIDGKMDKIREIVKSITDSEEEVVKLINHWRMLCINSSEMFGFNDGEEWMNSHVLFKKKKQI ScaNMT2MEMIADLLKRLEAGLVPDDEIRSLIRVELERRLKWGYKSTHQEQLDQ 84LLNLAHSIKKMKIASTEMDGLTSTMYEVPISLVQIQLGSHLKESCLYFKDETTTVDEAEIAMMDLYLERAQIKDGQSILDLGCGLGSVCFHIARKYTSCNITAVTNSVSQKEFIEEKSKTLNVPNVKVLLADITTLEMDDTFDCLFAIGLIEHMENYELLLRKLSDWMKQDGLLFIDHVCHKTLSYHFEPMDEDDWYTNLLFPAGTLTLVSASFLLYFQDDLSLVDHWSMSGKHFSRTNKEWLKNIDGKMDKIREIVKSITDSEEEVVKLINHWRMLdNSSE MFGFNDGEEWMNSHVLFKKKKQIPbrNMT2 MCTTMDTTKISQQDDLWKNMELGLISDEEVRRLMKIETEKRIKWGT 85KPTQQEQLAQLLDFNKSLRGMKMATEVHALENHKIYEIPDSFNQIIGGKESAGLFTDEATTTIEEANTKMMDLYCERAGLKDGQTILDIGCGAGLLVLHLAKKYKNCKITGVTNTSWHKEHILEQCKNLNLSNVEVILADVTTVDIERTFDRVFVIGLIEHMKNFELFLRKISKWMKDDGLLFLEHLCHKSFSDHWEPLSEDDWYAKNFFPSGTLVIPSATCLLYFQEDVTVKDHWLLSGNNFARSNEAILKRIDSKIEEVKDIFMSFYGIGEEEAVKLINWWRLLCITANELFKYNNGEEWLISQLLFKKKLMTCI PbrNMT1MVKGDQFQTTTMEETKISQENDLWTNMELGLIPDEEVRRLMKIEIEK 86RIEWGMKPTQHQQLAQLLDFTKSLRGMKMATELDKLDSKLYETPHSFNQIVNGSTLKESSGLYTDVTTTMDEASIKMMDLYCERANIKDGQTILDLGCGPGPLVLHIAKKYSNCKITGVTNAFSQREYILEECKKLSLSNVEIILADVTSLDLETTFDRVFVIGFIEHMKNFELFLRKISKWMKDDAVLFLEHFCHKSFSYHGEPLSEDDWYAKNFFAPGTLVIPSATCLLYFQEDLAVIDHWFLSGNHFARTNEEMLKGIDGKIEEIKDIFMSFYGINEAEAVKLINWWRLFCITGAEMFSYNNGEEWFISQLLFKKK EcaNMT4MALEQEDSMSVPERNEGVADLIKRMELGLVNDEEIRRLMRIQIENR 87LKWGYKPTHDQQLAQHLHFINSLKEMKMATEMDSLDSQVYESPNSFQQIMCGRSMKESAGLFMDDVTTVEEAHIRMMDLYCDKATFEDGQKILDLGCGHGSVVLHVAQKYKGCQVTGVTNSSAQKQYILEQCKKLDLSNVEIILADVTTLEMEEKFDRVIIIGLIEHMKNFKLFFQKVSKWMKEGGLLFLENYFHKDFAYHCEKIDEDDWYDGYIFPPGSLLMPSASTLLYFQEDLTVADHWVLPGTHFAKTFEEFLKKIDLRIEEVREIFEAFYGISKEEAMKLSNYWRNFCISAMEIFNYNNGQEWMISHLLYTKK CmaNMT5METGKNNQNMKTTIDDLWNQMMLGIVPDKEIRRLMKIELKKRLDW 88GYRPTHQQQLSQLLDFAKGLCNYCWTALRCMKMSAEFDTLDSKVYETPKSFQQIMCGTTIKESSGLFMNESTTLDQAQISMLDLYFDKAKIKDGQSILDLGCGHGALILYLAQKYQNCNITGVTNSLSQKEFIVEKCKKLGLSNVEILLADVTKLEMEDMFDRVFVIGLIEHMKNFELFLRKISEWMKPDGLLFLEHYCHKSFAHQWEPIDEEDWFSKYIFPPGTVIIPSASFLLYFQEDVKVIDHWTLSGNHFARTQEEWLKGIDGHIDEVEKTFESFYGISKEEAVKLINFWRVFCLSGVEMFGYNNGEEWMISHLLFKKK GflNMT4MTMEANNAKKEAIENLWEQMMMGLVPDHEITRLMKSELQKRLNWG 89YKPTHQQQISQLLDFAKSLRRMEMSLDFDNLELDTKMYETPESFQLIMSGTTLKESSGLFTDETATLDQTQIRMMDLYLEKAKIKDGQSILDLGCGHGALILHVAQKYRNCNVTGVTNSIAQKEFIFKQCKKLGLSNVEMVLADVTKCEMKATFDHIFVIGLIEHMKNFELFLRKVSEWMKSDGLLFMEHYCHKSFAYQWEPMDDDDLFSKYVFPPGSAIIPSASFLLYFQDDLTVVDHWTLSGNHFARTHQEWLKRIDSQSDEIKGIFESFYGISKEEAVKLINYWRVFCLFGVEMFGYNNGEEWMISHLLFKKK CchNMTSMEVVATSSARNPKKEIVDLWKRMELGLIPDEEIRDLMKIGLEKRLK 90WGYKPTHEQQLSQLLHFAKSLRSMKMASEMETLDDQMYETPTAFQQLMCGSTIKESAGFFKDESTTLDEAEIKMLDLYCEKARIEDGQKILDLGCGHGAVMLHIAQKYKNCNVTGVTNSISQQQFIVQRSKELNLSNVNMILADVTMLEMDATYDRIFIIGLIEHMKNFELFLRKISKWITKEGLLFLEHYCHKTFAYQCEPVDEDDWYNMFIFPPGTLILPSASFLLYFQDDLIVVDRWTLNGNHYARTQEEWLKRIDANVDGVKQMFESVCDGNKEEAVKLMNFWRIFCISGAEMLAYNNGEEWMISHYLFKKRN NsNMT2MEATQITKKQGVAELIKRIENGQVPDEEITRMMKIQIQKRLKLGYKS 91THEQQLAQLLHFVHSLQKMEMAEEVDTLDSELYEIPLPFLHIMCGKALKFSPGYFKDESTTLDESEVNMLDLYCERAQIEDGQTILDLGCGHGSLTLHVAKKYRGCKVTGITNSVSQKDFIMEECKKLNLSNVEIILEDVTKFETGTTYDRIFAVALIEHMKNYELFLKKVSAWMAQDGLLFVEHHCHKVFAYKYEPIDDDDWYTEYIFPTGTLVMSSSSILLYFQEDVSVVNHWTLSGKHPSLGFKQWLKRIDDNIDEIKEIFESFYGSKEKATKFITYWRVFCIAHSEMYATNGGEEWMLSQVLFKRK ScaNMT5MGGVADLLKKMELGLVPEEEIRRLMRIIIEKRLEWGYKPTHAEQLDH 92LTNFIQCLRGMKMADEIDALDAKMYEIPLPFMQTICGSTLKFSPGYFKDESTTLDESEIHMMDLYCERAEVKDGHSILDLGCGHGGFVLHVAQKYKNSIVTGVTNSVAEKEFIMTQCKKLCLSNVEIILADVTKFEPETTYDRVFAIALIEHMKNYELVLEKLSKWVAQDGFLFVEHHCHKVFPYKYEPLDEDDWYTEYIFPGGTIVLPSASILLYFQKDVSVVNHWSLNGKHPARGFKEWLKRLDENMDAVKAIFEPFYGSKEEAMKWITYWRVFCITH SEMYAYNNGEEWMLSQVLFKRKJdiNMT1 MSKGVAKLVERMELGLVSDDEVRRLMRILIEKRLKWGYKPTHEEQLT 93YLTNFIQGLKGMKIAEEIDALDAKMYEIPIAFMQILCGYSLKFSPGFFEDESTTLDESETIMMDLYCERAQVQDGQSILDLGCGHGGFVLHVAQKYKNCKVTGVTNSVSETEYIMEQCKKLGLSNVEIIIADVTKFEPEVTYDRVFAIALIEHMKNYELVLQKLSKWVAQDGFLFVDHHCHKVFPYKYEPIDEDDWYTQYIFPGGTLVLPSASILLYFQEDVSIVNHWTLSGNHPARGFKEWLKRLDDNMDEIKAIFEPFYGSKEEAMKWITYWRVFCITH SEMYAYNGGEEWMISQVLFKRKBthNMT1 MEVKQAGKEGVTELLVKRMELGLVPEEEIRRLMRIQIQKRLDWGYKP 94THEEQLAHLTKFIQNIRGMKMADEIDALDAKMYEIPLPFLQTICGKTLKFSPGYFKDESTTLDESETLMMDLYCERAQVKDGQSILDLGCGHGGFVLHLAQKYRNSVVTGVTNSVSETEYIKEQCKKLGLSNVEIIIADVTKFEPEVTYDRVFAIALIEHMKNYALVLNKISKWVAQDGYLFVEHHCHKVFPYKYEPLDEDDWYTNYIFPGGTLILPSASILLYFQEDVTVLNHWSLSGKHPSRGFIEWLKRLDENIDVIMGIFEPFYGSKEEATKWINYWRVFCMTHSEMYAYGNGEEWMLSQVLLKRK MaqNMT3MELGLVPEKEIRRLMRIQIQKRLEWGYKPTHEEQLAHLTKFIQNIRGM 95KMADEIDALDAKMYEIPLPFLQTICGKTLKFSPGYFKDESTTLDESETLMMDLYCERAQVKDGQSILDLGCGHGGFVLHLAQKYRNSIVTGVTNSVSETEYIKEQCKKLGLSNVEIIIADVTKFEPEVTYDRVFAIALIEHMKNYALVLNKISKWVAQDGYLFVEHHCHKVFPYKYEPLDEDDWYTNYIFPGGTLILPSASILLYFQEDVTVLNHWSLSGKHPSRGFIEWLKRLDENIDVIMGIFEPFYGSKEEATKWINYWRVFCITHSEMYAYGNGEEW MLSQVLLKRK McaNMT4MDKANERELKRAELFKKLEDDLVTYDEIKQVMRTELAKRLEWGYKP 96THQQQLAHLLDFAHALEGMKIANEVETLASEVYETPLPFXEIVLGPAKKXSSCLFEDESTTLEQAEIAMLDLYFERAQIRXGMSVLDLGCGXGSVGLHIARKYKNCXVTCITNSISQKQYIENQCKLYNLSNVKIILADIVAHDTDDTFDVVLVIGVIEHMKNYALLLNKISKWMAKDGLLFVEHLCHKTFPYHFEPLDEDDWYSNFVFPTGTLTMPSVSFLLYFQADVSILNHWILSGKNFSRTXEEFLKRIDANVDAIKDGLKPSLGSEGVAKLISYWRGFCLTGMEMFGYNNGEEWMVSQVLFKNK TcoNMT3MEDNNNLLQEEMNVVELLQRPELGLVPDEKIRKLTRLQLQKRLKWG 97YKPTHEAQLSHLFQFIHSLPSLNMESEDENPKSWLYETPTSFLQLLYGDCIKESDTYYKEDTATLEEAVINMLELYCERARITEGLSVLDLGCGYGALTLHVAQKYKSCKVTGVTSSISQKQYIMEKCKKLNLTNVEIILADVATIEIEAASYDRIFALGIFEHVNDYKLFLGKLSKWMKQDGLLFVEYLCHKTFPYQNKPLDKGDKWYNEYVFPSGGLIIPSASFILYFQNDVSVVRQWTQGGQHSARTFEELLKRIDGNIDKIKEIFIESYGSKEDAVRFINYWRVFLITGVEMFSYNDGEEWMGAHFLFKKKFIMQE CmuNMT4MEVKQSKGDELRSRVAELLERPELGLVPDEEIRRLAKARLEKRLKW 98GYKATHGEQLSSLLQFVESLPSLNMASEDDSPKAWLYETPTSFLQLIYGDIIKESGSYYKDESTTLEEAMIHNMNLCCERANIKEGQSVVDLGCGYGAFILHVAQKYKTCRVTGITSSISQKHYIMEQCKKLNLSNVEVILADVATIKLDATFDRVFAAGMFEHVNDYKSFLRKITNWMKPDGRLFVEHLCNKTFPYQNKPLDDGDNWGEYVFPSGGLIIPSASLLLYFQEDVSIVNHWTFSGKHAANKFEELLKRIDAKIDAIKRIFNECYGSKDSIRFINYWRVFLITAAEMFGYNNGEEWMGVHLLFKKK CtrNMT2GLKSSVAELLERPELGLVPDGEIRKLTKTRLAKRLEWGYKATHEDQLS 99HLLRFIHSLPSLNMASEDDSPKAWLYETPTSFLQLIYGDIIKESGTYYKDESSTLEEAIIHNMDLCCERARIKEGQSVLDLGCGYGAFTLHVAQKYKSCSVTGITSSISQKDYIMEQCKKLNLSNVEVILADVATIKMNTTFDRVFALGMFEHINDYKLFLRRISNWMKHDGLLFVEHLCNKTFAYQNKPLDDGDDWFNEYVFPSAGLIIPSASLLLYFQEDVSIVHHWTFSGKHAAYKFEELLERIDAKIEAIKEIFIECYGSKEDAIRFINYWRVFLITAA EMFAYRDGEEWMGSHVLFKKKCmuNMT5 MEAKQHESNNNIDEELKNRVNIGEQEERPGFEDEEIRRLAKAQLAKR 100LKWGYKPTHEQQLSHLLQFLQSLPSLNMASEDESSKAWLYETPTSFLQLLFGNVIKFSGYYYKHESSTFEESMIHNMDLCCERANIKEGQNVIDLGCGYGAFVLHVAQKYKSCSVTGITCSITQKHHIMEECKKLNLCNVKVILADVATIELGTAFDRVFAFGMFEEINDYKLILRKISNWMKPDGLFFVEHLCHKTLAYQNKLIDDQDWYEEYIFPSGGLIVPSASLLLYFQDDLSVVYHWTYNGKHGARSFEKMLERTDANIDTIKDMFTEFYGSKEKAIKFINYWRVFFITAAEMFAYNDGEEWMCSQLLFKKK CmuNMT8MEHKIEDIRKLKSRVEEQLERPELGLVKDEDIKTLAKAKLEKRLKWG 101YKPTYAEQLSNLLQFAQSLPSLKMENVDDQGSSKQWLYGVPSEFLQIIYGGIIKMSGSYYEDESTTLEESMIKDMDSCCEKANVKEGHSVLDIGCGYGSLIIHIAKKYRTCNVTGITNFVEQKQYIMEECKKLNLSNVEVIVGDGTTINLNTTTFDRVFVTGMLEEINDYKLFLKSVSDWMKPDGLLLVTHFCHKTFAYQNNKALDDEDWHNEYIFPSGNLIVPSASLLLYFQEDLSVVSHWATNGTHTGRTCKKLVERIDANIEKIKEIFSEFYGSKEDAIRMINYWRVLCITGAEMYTCKDGEEWMDVYYLFKKK

TABLE 6 Variants of BM3 N-demethylase BM3 SEQ ID variant NO: Genotype8F11 L437A 4H9 L181A, T260A, L437A 8C7 L75A, L181A 4H5L75A, M177A, L181A 7A1 L75A, M177A, L181A, T260A Amino Acid Sequence8F11 MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEA 102PGRVTRYLSSQRLIKEACDESRFDKNLSQALKFARDFAGDGLVTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVSEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFIISMVRALDEVMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKARGEQSDDLLTQMLNGKDPETGEPLDDGNIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKVAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDEVMVLIPQLHRDKTVWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETATLKPKGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG 4H9MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEA 103PGRVTRYLSSQRLIKEACDESRFDKNLSQALKFARDFAGDGLVTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVSEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFIISMVRAADEVMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKARGEQSDDLLTQMLNGKDPETGEPLDDGNIRYQIIAFLIAGHETTSGLLSFALYFLVKNPHVLQKVAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDEVMVLIPQLHRDKTVWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETATLKPKGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG 8C7MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEA 104PGRVTRYLSSQRLIKEACDESRFDKNLSQAAKFARDFAGDGLVTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVSEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFIISMVRAADEVMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKARGEQSDDLLTQMLNGKDPETGEPLDDGNIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKVAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDEVMVLIPQLHRDKTVWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPKGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG 4H5MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEA 105PGRVTRYLSSQRLIKEACDESRFDKNLSQAAKFARDFAGDGLVTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVSEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFIISAVRAADEVMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKARGEQSDDLLTQMLNGKDPETGEPLDDGNIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKVAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDEVMVLIPQLHRDKTVWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPKGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG 7A1MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEA 106PGRVTRYLSSQRLIKEACDESRFDKNLSQAAKFARDFAGDGLVTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVSEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFIISAVRAADEVMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKARGEQSDDLLTQMLNGKDPETGEPLDDGNIRYQIIAFLIAGHETTSGLLSFALYFLVKNPHVLQKVAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDEVMVLIPQLHRDKTVWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPKGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG Nucleotide Sequence 8F11ATGACCATCAAAGAAATGCCACAACCTAAGACTTTCGGTGAA 107TTGAAGAATTTGCCTTTGTTGAACACCGATAAGCCAGTTCAAGCTTTGATGAAGATTGCTGATGAATTGGGTGAAATCTTCAAGTTTGAAGCTCCAGGTAGAGTCACTAGATACTTGTCATCTCAAAGATTGATCAAAGAAGCCTGCGACGAATCCAGATTTGATAAGAATTTGTCTCAAGCTTTGAAGTTCGCTAGAGATTTTGCTGGTGATGGTTTGGTTACTTCTTGGACTCACGAAAAGAATTGGAAGAAGGCCCATAACATTTTGTTGCCATCTTTCTCACAACAAGCCATGAAGGGTTATCATGCTATGATGGTTGATATCGCCGTTCAATTGGTTCAAAAGTGGGAAAGATTGAACGCCGATGAACATATCGAAGTCTCTGAAGATATGACCAGATTGACCTTGGATACCATTGGTTTGTGTGGTTTCAACTACAGATTCAACTCCTTCTACAGAGATCAACCACATCCATTCATCATCTCTATGGTTAGAGCTTTGGATGAAGTCATGAACAAATTGCAAAGAGCTAATCCAGACGATCCAGCTTATGACGAAAACAAGAGACAATTCCAAGAAGATATCAAGGTCATGAACGATTTGGTCGATAAGATTATCGCTGATAGAAAGGCTAGAGGTGAACAATCTGATGATTTGTTGACCCAAATGTTGAACGGTAAGGATCCAGAAACTGGTGAACCATTGGATGATGGTAACATCAGATACCAAATTATCACCTTCTTGATTGCTGGTCACGAAACTACATCTGGTTTGTTGTCTTTTGCCTTGTACTTTTTGGTTAAGAACCCACACGTCTTGCAAAAGGTTGCTGAAGAAGCTGCAAGAGTTTTGGTTGATCCAGTTCCATCTTACAAGCAAGTCAAGCAATTGAAGTACGTTGGTATGGTTTTGAACGAAGCTTTGAGATTGTGGCCAACTGCTCCAGCTTTTTCATTATACGCTAAAGAAGATACCGTCTTGGGTGGTGAATATCCATTGGAAAAAGGTGATGAAGTTATGGTCTTGATCCCACAATTGCATAGAGATAAGACTGTTTGGGGTGATGATGTCGAAGAATTCAGACCAGAAAGATTCGAAAACCCATCTGCTATTCCACAACATGCTTTTAAGCCATTTGGTAACGGTCAAAGAGCTTGCATTGGTCAACAATTCGCTTTACATGAAGCTACCTTGGTTTTGGGTATGATGTTGAAACACTTCGACTTCGAAGATCACACCAACTACGAATTGGATATCAAAGAAACCGCTACCTTGAAGCCAAAGGGTTTTGTTGTTAAGGCTAAGTCCAAAAAGATTCCATTGGGTGGTATTCCATCTCCATCTACTGAACAATCCGCTAAGAAGGTTAGAAAGAAAGCTGAAAACGCTCATAACACACCTTTGTTGGTCTTGTACGGTTCTAATATGGGTACTGCTGAAGGTACAGCAAGAGATTTGGCAGATATTGCTATGTCTAAAGGTTTCGCTCCACAAGTTGCTACTTTGGATTCTCATGCTGGTAATTTGCCAAGAGAAGGTGCTGTTTTGATAGTTACTGCTTCTTACAATGGTCACCCACCAGATAATGCTAAGCAATTCGTTGATTGGTTGGATCAAGCTTCAGCTGATGAAGTAAAAGGTGTTAGATACTCTGTTTTCGGTTGCGGTGACAAAAATTGGGCTACTACTTATCAAAAGGTTCCAGCCTTTATTGACGAAACTTTGGCTGCTAAAGGTGCTGAAAACATTGCTGACAGAGGTGAAGCTGATGCCTCCGACGACTTCGAAGGTACTTACGAAGAATGGAGAGAACACATGTGGTCTGACGTTGCTGCTTACTTCAACTTGGACATCGAAAACTCTGAAGACAACAAGTCCACTTTGTCTTTGCAATTCGTTGACTCCGCTGCTGACATGCCATTGGCTAAGATGCACGGTGCTTTCTCTACCAACGTCGTTGCCTCCAAGGAATTGCAACAACCAGGTTCTGCTAGATCTACTAGACACTTGGAAATCGAATTGCCAAAGGAAGCTTCCTACCAAGAAGGTGACCACTTGGGCGTTATTCCAAGAAACTACGAAGGTATCGTCAACAGAGTTACTGCTAGATTCGGTTTGGATGCTTCTCAACAAATCAGATTAGAAGCTGAAGAAGAAAAGTTGGCTCACTTGCCATTAGCTAAGACTGTCTCCGTTGAAGAATTGTTGCAATACGTCGAATTGCAAGACCCAGTTACCAGAACCCAATTGAGAGCCATGGCTGCCAAGACCGTCTGTCCACCACACAAGGTTGAATTGGAAGCCTTGTTGGAAAAGCAAGCCTACAAGGAACAAGTTTTGGCTAAGAGATTGACCATGTTGGAATTGTTGGAAAAGTACCCAGCCTGCGAAATGAAGTTCTCTGAATTTATCGCCTTGTTGCCATCTATCAGACCACGTTACTACTCTATTTCTTCCTCTCCACGTGTTGACGAAAAGCAAGCTTCTATTACTGTTTCCGTTGTCTCCGGTGAAGCTTGGTCCGGTTACGGTGAATACAAGGGTATTGCTTCTAACTACTTGGCTGAATTGCAAGAAGGTGACACCATTACTTGTTTCATCTCTACTCCACAATCCGAATTTACTTTGCCAAAGGACCCAGAAACTCCATTGATCATGGTTGGTCCAGGTACTGGTGTCGCTCCATTCAGAGGTTTCGTTCAAGCTAGAAAACAATTGAAGGAACAAGGTCAATCTTTGGGTGAAGCTCACTTGTACTTCGGTTGTAGATCTCCACACGAAGACTACTTATACCAAGAAGAATTGGAAAACGCTCAATCCGAAGGTATTATCACTTTGCACACCGCTTTCTCCAGAATGCCAAACCAACCAAAGACTTACGTCCAACACGTTATGGAACAAGACGGTAAGAAGTTGATTGAATTGTTGGACCAAGGTGCTCACTTCTACATTTGTGGTGATGGTTCTCAAATGGCTCCAGCCGTTGAAGCCACTTTGATGAAGTCTTACGCTGATGTTCACCAAGTTTCCGAAGCCGATGCTAGATTATGGTTGCAACAATTGGAAGAAAAAGGTCGTTAC GCTAAGGATGTCTGGGCCGGTTGA 4H9ATGACCATCAAAGAAATGCCACAACCTAAGACTTTCGGTGAA 108TTGAAGAATTTGCCTTTGTTGAACACCGATAAGCCAGTTCAAGCTTTGATGAAGATTGCTGATGAATTGGGTGAAATCTTCAAGTTTGAAGCTCCAGGTAGAGTCACTAGATACTTGTCATCTCAAAGATTGATCAAAGAAGCCTGCGACGAATCCAGATTTGATAAGAATTTGTCTCAAGCTTTGAAGTTCGCTAGAGATTTTGCTGGTGATGGTTTGGTTACTTCTTGGACTCACGAAAAGAATTGGAAGAAGGCCCATAACATTTTGTTGCCATCTTTCTCACAACAAGCCATGAAGGGTTATCATGCTATGATGGTTGATATCGCCGTTCAATTGGTTCAAAAGTGGGAAAGATTGAACGCCGATGAACATATCGAAGTCTCTGAAGATATGACCAGATTGACCTTGGATACCATTGGTTTGTGTGGTTTCAACTACAGATTCAACTCCTTCTACAGAGATCAACCACATCCATTCATCATCTCTATGGTTAGAGCTGCAGATGAAGTCATGAACAAATTGCAAAGAGCTAATCCAGACGATCCAGCTTATGACGAAAACAAGAGACAATTCCAAGAAGATATCAAGGTCATGAACGATTTGGTCGATAAGATTATCGCTGATAGAAAGGCTAGAGGTGAACAATCTGATGATTTGTTGACCCAAATGTTGAACGGTAAGGATCCAGAAACTGGTGAACCATTGGATGATGGTAACATCAGATACCAAATTATCGCTTTCTTGATTGCTGGTCACGAAACTACATCTGGTTTGTTGTCTTTTGCCTTGTACTTTTTGGTTAAGAACCCACACGTCTTGCAAAAGGTTGCTGAAGAAGCTGCAAGAGTTTTGGTTGATCCAGTTCCATCTTACAAGCAAGTCAAGCAATTGAAGTACGTTGGTATGGTTTTGAACGAAGCTTTGAGATTGTGGCCAACTGCTCCAGCTTTTTCATTATACGCTAAAGAAGATACCGTCTTGGGTGGTGAATATCCATTGGAAAAAGGTGATGAAGTTATGGTCTTGATCCCACAATTGCATAGAGATAAGACTGTTTGGGGTGATGATGTCGAAGAATTCAGACCAGAAAGATTCGAAAACCCATCTGCTATTCCACAACATGCTTTTAAGCCATTTGGTAACGGTCAAAGAGCTTGCATTGGTCAACAATTCGCTTTACATGAAGCTACCTTGGTTTTGGGTATGATGTTGAAACACTTCGACTTCGAAGATCACACCAACTACGAATTGGATATCAAAGAAACCGCTACCTTGAAGCCAAAGGGTTTTGTTGTTAAGGCTAAGTCCAAAAAGATTCCATTGGGTGGTATTCCATCTCCATCTACTGAACAATCCGCTAAGAAGGTTAGAAAGAAAGCTGAAAACGCTCATAACACACCTTTGTTGGTCTTGTACGGTTCTAATATGGGTACTGCTGAAGGTACAGCAAGAGATTTGGCAGATATTGCTATGTCTAAAGGTTTCGCTCCACAAGTTGCTACTTTGGATTCTCATGCTGGTAATTTGCCAAGAGAAGGTGCTGTTTTGATAGTTACTGCTTCTTACAATGGTCACCCACCAGATAATGCTAAGCAATTCGTTGATTGGTTGGATCAAGCTTCAGCTGATGAAGTAAAAGGTGTTAGATACTCTGTTTTCGGTTGCGGTGACAAAAATTGGGCTACTACTTATCAAAAGGTTCCAGCCTTTATTGACGAAACTTTGGCTGCTAAAGGTGCTGAAAACATTGCTGACAGAGGTGAAGCTGATGCCTCCGACGACTTCGAAGGTACTTACGAAGAATGGAGAGAACACATGTGGTCTGACGTTGCTGCTTACTTCAACTTGGACATCGAAAACTCTGAAGACAACAAGTCCACTTTGTCTTTGCAATTCGTTGACTCCGCTGCTGACATGCCATTGGCTAAGATGCACGGTGCTTTCTCTACCAACGTCGTTGCCTCCAAGGAATTGCAACAACCAGGTTCTGCTAGATCTACTAGACACTTGGAAATCGAATTGCCAAAGGAAGCTTCCTACCAAGAAGGTGACCACTTGGGCGTTATTCCAAGAAACTACGAAGGTATCGTCAACAGAGTTACTGCTAGATTCGGTTTGGATGCTTCTCAACAAATCAGATTAGAAGCTGAAGAAGAAAAGTTGGCTCACTTGCCATTAGCTAAGACTGTCTCCGTTGAAGAATTGTTGCAATACGTCGAATTGCAAGACCCAGTTACCAGAACCCAATTGAGAGCCATGGCTGCCAAGACCGTCTGTCCACCACACAAGGTTGAATTGGAAGCCTTGTTGGAAAAGCAAGCCTACAAGGAACAAGTTTTGGCTAAGAGATTGACCATGTTGGAATTGTTGGAAAAGTACCCAGCCTGCGAAATGAAGTTCTCTGAATTTATCGCCTTGTTGCCATCTATCAGACCACGTTACTACTCTATTTCTTCCTCTCCACGTGTTGACGAAAAGCAAGCTTCTATTACTGTTTCCGTTGTCTCCGGTGAAGCTTGGTCCGGTTACGGTGAATACAAGGGTATTGCTTCTAACTACTTGGCTGAATTGCAAGAAGGTGACACCATTACTTGTTTCATCTCTACTCCACAATCCGAATTTACTTTGCCAAAGGACCCAGAAACTCCATTGATCATGGTTGGTCCAGGTACTGGTGTCGCTCCATTCAGAGGTTTCGTTCAAGCTAGAAAACAATTGAAGGAACAAGGTCAATCTTTGGGTGAAGCTCACTTGTACTTCGGTTGTAGATCTCCACACGAAGACTACTTATACCAAGAAGAATTGGAAAACGCTCAATCCGAAGGTATTATCACTTTGCACACCGCTTTCTCCAGAATGCCAAACCAACCAAAGACTTACGTCCAACACGTTATGGAACAAGACGGTAAGAAGTTGATTGAATTGTTGGACCAAGGTGCTCACTTCTACATTTGTGGTGATGGTTCTCAAATGGCTCCAGCCGTTGAAGCCACTTTGATGAAGTCTTACGCTGATGTTCACCAAGTTTCCGAAGCCGATGCTAGATTATGGTTGCAACAATTGGAAGAAAAAGGTCGTTAC GCTAAGGATGTCTGGGCCGGTTGA 8C7ATGACCATCAAAGAAATGCCACAACCTAAGACTTTCGGTGAA 109TTGAAGAATTTGCCTTTGTTGAACACCGATAAGCCAGTTCAAGCTTTGATGAAGATTGCTGATGAATTGGGTGAAATCTTCAAGTTTGAAGCTCCAGGTAGAGTCACTAGATACTTGTCATCTCAAAGATTGATCAAAGAAGCCTGCGACGAATCCAGATTTGATAAGAATTTGTCTCAAGCTGCTAAGTTCGCTAGAGATTTTGCTGGTGATGGTTTGGTTACTTCTTGGACTCACGAAAAGAATTGGAAGAAGGCCCATAACATTTTGTTGCCATCTTTCTCACAACAAGCCATGAAGGGTTATCATGCTATGATGGTTGATATCGCCGTTCAATTGGTTCAAAAGTGGGAAAGATTGAACGCCGATGAACATATCGAAGTCTCTGAAGATATGACCAGATTGACCTTGGATACCATTGGTTTGTGTGGTTTCAACTACAGATTCAACTCCTTCTACAGAGATCAACCACATCCATTCATCATCTCTATGGTTAGAGCTGCAGATGAAGTCATGAACAAATTGCAAAGAGCTAATCCAGACGATCCAGCTTATGACGAAAACAAGAGACAATTCCAAGAAGATATCAAGGTCATGAACGATTTGGTCGATAAGATTATCGCTGATAGAAAGGCTAGAGGTGAACAATCTGATGATTTGTTGACCCAAATGTTGAACGGTAAGGATCCAGAAACTGGTGAACCATTGGATGATGGTAACATCAGATACCAAATTATCACCTTCTTGATTGCTGGTCACGAAACTACATCTGGTTTGTTGTCTTTTGCCTTGTACTTTTTGGTTAAGAACCCACACGTCTTGCAAAAGGTTGCTGAAGAAGCTGCAAGAGTTTTGGTTGATCCAGTTCCATCTTACAAGCAAGTCAAGCAATTGAAGTACGTTGGTATGGTTTTGAACGAAGCTTTGAGATTGTGGCCAACTGCTCCAGCTTTTTCATTATACGCTAAAGAAGATACCGTCTTGGGTGGTGAATATCCATTGGAAAAAGGTGATGAAGTTATGGTCTTGATCCCACAATTGCATAGAGATAAGACTGTTTGGGGTGATGATGTCGAAGAATTCAGACCAGAAAGATTCGAAAACCCATCTGCTATTCCACAACATGCTTTTAAGCCATTTGGTAACGGTCAAAGAGCTTGCATTGGTCAACAATTCGCTTTACATGAAGCTACCTTGGTTTTGGGTATGATGTTGAAACACTTCGACTTCGAAGATCACACCAACTACGAATTGGATATCAAAGAAACCTTGACCTTGAAGCCAAAGGGTTTTGTTGTTAAGGCTAAGTCCAAAAAGATTCCATTGGGTGGTATTCCATCTCCATCTACTGAACAATCCGCTAAGAAGGTTAGAAAGAAAGCTGAAAACGCTCATAACACACCTTTGTTGGTCTTGTACGGTTCTAATATGGGTACTGCTGAAGGTACAGCAAGAGATTTGGCAGATATTGCTATGTCTAAAGGTTTCGCTCCACAAGTTGCTACTTTGGATTCTCATGCTGGTAATTTGCCAAGAGAAGGTGCTGTTTTGATAGTTACTGCTTCTTACAATGGTCACCCACCAGATAATGCTAAGCAATTCGTTGATTGGTTGGATCAAGCTTCAGCTGATGAAGTAAAAGGTGTTAGATACTCTGTTTTCGGTTGCGGTGACAAAAATTGGGCTACTACTTATCAAAAGGTTCCAGCCTTTATTGACGAAACTTTGGCTGCTAAAGGTGCTGAAAACATTGCTGACAGAGGTGAAGCTGATGCCTCCGACGACTTCGAAGGTACTTACGAAGAATGGAGAGAACACATGTGGTCTGACGTTGCTGCTTACTTCAACTTGGACATCGAAAACTCTGAAGACAACAAGTCCACTTTGTCTTTGCAATTCGTTGACTCCGCTGCTGACATGCCATTGGCTAAGATGCACGGTGCTTTCTCTACCAACGTCGTTGCCTCCAAGGAATTGCAACAACCAGGTTCTGCTAGATCTACTAGACACTTGGAAATCGAATTGCCAAAGGAAGCTTCCTACCAAGAAGGTGACCACTTGGGCGTTATTCCAAGAAACTACGAAGGTATCGTCAACAGAGTTACTGCTAGATTCGGTTTGGATGCTTCTCAACAAATCAGATTAGAAGCTGAAGAAGAAAAGTTGGCTCACTTGCCATTAGCTAAGACTGTCTCCGTTGAAGAATTGTTGCAATACGTCGAATTGCAAGACCCAGTTACCAGAACCCAATTGAGAGCCATGGCTGCCAAGACCGTCTGTCCACCACACAAGGTTGAATTGGAAGCCTTGTTGGAAAAGCAAGCCTACAAGGAACAAGTTTTGGCTAAGAGATTGACCATGTTGGAATTGTTGGAAAAGTACCCAGCCTGCGAAATGAAGTTCTCTGAATTTATCGCCTTGTTGCCATCTATCAGACCACGTTACTACTCTATTTCTTCCTCTCCACGTGTTGACGAAAAGCAAGCTTCTATTACTGTTTCCGTTGTCTCCGGTGAAGCTTGGTCCGGTTACGGTGAATACAAGGGTATTGCTTCTAACTACTTGGCTGAATTGCAAGAAGGTGACACCATTACTTGTTTCATCTCTACTCCACAATCCGAATTTACTTTGCCAAAGGACCCAGAAACTCCATTGATCATGGTTGGTCCAGGTACTGGTGTCGCTCCATTCAGAGGTTTCGTTCAAGCTAGAAAACAATTGAAGGAACAAGGTCAATCTTTGGGTGAAGCTCACTTGTACTTCGGTTGTAGATCTCCACACGAAGACTACTTATACCAAGAAGAATTGGAAAACGCTCAATCCGAAGGTATTATCACTTTGCACACCGCTTTCTCCAGAATGCCAAACCAACCAAAGACTTACGTCCAACACGTTATGGAACAAGACGGTAAGAAGTTGATTGAATTGTTGGACCAAGGTGCTCACTTCTACATTTGTGGTGATGGTTCTCAAATGGCTCCAGCCGTTGAAGCCACTTTGATGAAGTCTTACGCTGATGTTCACCAAGTTTCCGAAGCCGATGCTAGATTATGGTTGCAACAATTGGAAGAAAAAGGTCGTTAC GCTAAGGATGTCTGGGCCGGTTGA 4H5ATGACCATCAAAGAAATGCCACAACCTAAGACTTTCGGTGAA 110TTGAAGAATTTGCCTTTGTTGAACACCGATAAGCCAGTTCAAGCTTTGATGAAGATTGCTGATGAATTGGGTGAAATCTTCAAGTTTGAAGCTCCAGGTAGAGTCACTAGATACTTGTCATCTCAAAGATTGATCAAAGAAGCCTGCGACGAATCCAGATTTGATAAGAATTTGTCTCAAGCTGCTAAGTTCGCTAGAGATTTTGCTGGTGATGGTTTGGTTACTTCTTGGACTCACGAAAAGAATTGGAAGAAGGCCCATAACATTTTGTTGCCATCTTTCTCACAACAAGCCATGAAGGGTTATCATGCTATGATGGTTGATATCGCCGTTCAATTGGTTCAAAAGTGGGAAAGATTGAACGCCGATGAACATATCGAAGTCTCTGAAGATATGACCAGATTGACCTTGGATACCATTGGTTTGTGTGGTTTCAACTACAGATTCAACTCCTTCTACAGAGATCAACCACATCCATTCATCATCTCTGCTGTTAGAGCTGCAGATGAAGTCATGAACAAATTGCAAAGAGCTAATCCAGACGATCCAGCTTATGACGAAAACAAGAGACAATTCCAAGAAGATATCAAGGTCATGAACGATTTGGTCGATAAGATTATCGCTGATAGAAAGGCTAGAGGTGAACAATCTGATGATTTGTTGACCCAAATGTTGAACGGTAAGGATCCAGAAACTGGTGAACCATTGGATGATGGTAACATCAGATACCAAATTATCACCTTCTTGATTGCTGGTCACGAAACTACATCTGGTTTGTTGTCTTTTGCCTTGTACTTTTTGGTTAAGAACCCACACGTCTTGCAAAAGGTTGCTGAAGAAGCTGCAAGAGTTTTGGTTGATCCAGTTCCATCTTACAAGCAAGTCAAGCAATTGAAGTACGTTGGTATGGTTTTGAACGAAGCTTTGAGATTGTGGCCAACTGCTCCAGCTTTTTCATTATACGCTAAAGAAGATACCGTCTTGGGTGGTGAATATCCATTGGAAAAAGGTGATGAAGTTATGGTCTTGATCCCACAATTGCATAGAGATAAGACTGTTTGGGGTGATGATGTCGAAGAATTCAGACCAGAAAGATTCGAAAACCCATCTGCTATTCCACAACATGCTTTTAAGCCATTTGGTAACGGTCAAAGAGCTTGCATTGGTCAACAATTCGCTTTACATGAAGCTACCTTGGTTTTGGGTATGATGTTGAAACACTTCGACTTCGAAGATCACACCAACTACGAATTGGATATCAAAGAAACCTTGACCTTGAAGCCAAAGGGTTTTGTTGTTAAGGCTAAGTCCAAAAAGATTCCATTGGGTGGTATTCCATCTCCATCTACTGAACAATCCGCTAAGAAGGTTAGAAAGAAAGCTGAAAACGCTCATAACACACCTTTGTTGGTCTTGTACGGTTCTAATATGGGTACTGCTGAAGGTACAGCAAGAGATTTGGCAGATATTGCTATGTCTAAAGGTTTCGCTCCACAAGTTGCTACTTTGGATTCTCATGCTGGTAATTTGCCAAGAGAAGGTGCTGTTTTGATAGTTACTGCTTCTTACAATGGTCACCCACCAGATAATGCTAAGCAATTCGTTGATTGGTTGGATCAAGCTTCAGCTGATGAAGTAAAAGGTGTTAGATACTCTGTTTTCGGTTGCGGTGACAAAAATTGGGCTACTACTTATCAAAAGGTTCCAGCCTTTATTGACGAAACTTTGGCTGCTAAAGGTGCTGAAAACATTGCTGACAGAGGTGAAGCTGATGCCTCCGACGACTTCGAAGGTACTTACGAAGAATGGAGAGAACACATGTGGTCTGACGTTGCTGCTTACTTCAACTTGGACATCGAAAACTCTGAAGACAACAAGTCCACTTTGTCTTTGCAATTCGTTGACTCCGCTGCTGACATGCCATTGGCTAAGATGCACGGTGCTTTCTCTACCAACGTCGTTGCCTCCAAGGAATTGCAACAACCAGGTTCTGCTAGATCTACTAGACACTTGGAAATCGAATTGCCAAAGGAAGCTTCCTACCAAGAAGGTGACCACTTGGGCGTTATTCCAAGAAACTACGAAGGTATCGTCAACAGAGTTACTGCTAGATTCGGTTTGGATGCTTCTCAACAAATCAGATTAGAAGCTGAAGAAGAAAAGTTGGCTCACTTGCCATTAGCTAAGACTGTCTCCGTTGAAGAATTGTTGCAATACGTCGAATTGCAAGACCCAGTTACCAGAACCCAATTGAGAGCCATGGCTGCCAAGACCGTCTGTCCACCACACAAGGTTGAATTGGAAGCCTTGTTGGAAAAGCAAGCCTACAAGGAACAAGTTTTGGCTAAGAGATTGACCATGTTGGAATTGTTGGAAAAGTACCCAGCCTGCGAAATGAAGTTCTCTGAATTTATCGCCTTGTTGCCATCTATCAGACCACGTTACTACTCTATTTCTTCCTCTCCACGTGTTGACGAAAAGCAAGCTTCTATTACTGTTTCCGTTGTCTCCGGTGAAGCTTGGTCCGGTTACGGTGAATACAAGGGTATTGCTTCTAACTACTTGGCTGAATTGCAAGAAGGTGACACCATTACTTGTTTCATCTCTACTCCACAATCCGAATTTACTTTGCCAAAGGACCCAGAAACTCCATTGATCATGGTTGGTCCAGGTACTGGTGTCGCTCCATTCAGAGGTTTCGTTCAAGCTAGAAAACAATTGAAGGAACAAGGTCAATCTTTGGGTGAAGCTCACTTGTACTTCGGTTGTAGATCTCCACACGAAGACTACTTATACCAAGAAGAATTGGAAAACGCTCAATCCGAAGGTATTATCACTTTGCACACCGCTTTCTCCAGAATGCCAAACCAACCAAAGACTTACGTCCAACACGTTATGGAACAAGACGGTAAGAAGTTGATTGAATTGTTGGACCAAGGTGCTCACTTCTACATTTGTGGTGATGGTTCTCAAATGGCTCCAGCCGTTGAAGCCACTTTGATGAAGTCTTACGCTGATGTTCACCAAGTTTCCGAAGCCGATGCTAGATTATGGTTGCAACAATTGGAAGAAAAAGGTCGTTAC GCTAAGGATGTCTGGGCCGGTTGA 7A1ATGACCATCAAAGAAATGCCACAACCTAAGACTTTCGGTGAA 111TTGAAGAATTTGCCTTTGTTGAACACCGATAAGCCAGTTCAAGCTTTGATGAAGATTGCTGATGAATTGGGTGAAATCTTCAAGTTTGAAGCTCCAGGTAGAGTCACTAGATACTTGTCATCTCAAAGATTGATCAAAGAAGCCTGCGACGAATCCAGATTTGATAAGAATTTGTCTCAAGCTGCTAAGTTCGCTAGAGATTTTGCTGGTGATGGTTTGGTTACTTCTTGGACTCACGAAAAGAATTGGAAGAAGGCCCATAACATTTTGTTGCCATCTTTCTCACAACAAGCCATGAAGGGTTATCATGCTATGATGGTTGATATCGCCGTTCAATTGGTTCAAAAGTGGGAAAGATTGAACGCCGATGAACATATCGAAGTCTCTGAAGATATGACCAGATTGACCTTGGATACCATTGGTTTGTGTGGTTTCAACTACAGATTCAACTCCTTCTACAGAGATCAACCACATCCATTCATCATCTCTGCTGTTAGAGCTGCAGATGAAGTCATGAACAAATTGCAAAGAGCTAATCCAGACGATCCAGCTTATGACGAAAACAAGAGACAATTCCAAGAAGATATCAAGGTCATGAACGATTTGGTCGATAAGATTATCGCTGATAGAAAGGCTAGAGGTGAACAATCTGATGATTTGTTGACCCAAATGTTGAACGGTAAGGATCCAGAAACTGGTGAACCATTGGATGATGGTAACATCAGATACCAAATTATCGCTTTCTTGATTGCTGGTCACGAAACTACATCTGGTTTGTTGTCTTTTGCCTTGTACTTTTTGGTTAAGAACCCACACGTCTTGCAAAAGGTTGCTGAAGAAGCTGCAAGAGTTTTGGTTGATCCAGTTCCATCTTACAAGCAAGTCAAGCAATTGAAGTACGTTGGTATGGTTTTGAACGAAGCTTTGAGATTGTGGCCAACTGCTCCAGCTTTTTCATTATACGCTAAAGAAGATACCGTCTTGGGTGGTGAATATCCATTGGAAAAAGGTGATGAAGTTATGGTCTTGATCCCACAATTGCATAGAGATAAGACTGTTTGGGGTGATGATGTCGAAGAATTCAGACCAGAAAGATTCGAAAACCCATCTGCTATTCCACAACATGCTTTTAAGCCATTTGGTAACGGTCAAAGAGCTTGCATTGGTCAACAATTCGCTTTACATGAAGCTACCTTGGTTTTGGGTATGATGTTGAAACACTTCGACTTCGAAGATCACACCAACTACGAATTGGATATCAAAGAAACCTTGACCTTGAAGCCAAAGGGTTTTGTTGTTAAGGCTAAGTCCAAAAAGATTCCATTGGGTGGTATTCCATCTCCATCTACTGAACAATCCGCTAAGAAGGTTAGAAAGAAAGCTGAAAACGCTCATAACACACCTTTGTTGGTCTTGTACGGTTCTAATATGGGTACTGCTGAAGGTACAGCAAGAGATTTGGCAGATATTGCTATGTCTAAAGGTTTCGCTCCACAAGTTGCTACTTTGGATTCTCATGCTGGTAATTTGCCAAGAGAAGGTGCTGTTTTGATAGTTACTGCTTCTTACAATGGTCACCCACCAGATAATGCTAAGCAATTCGTTGATTGGTTGGATCAAGCTTCAGCTGATGAAGTAAAAGGTGTTAGATACTCTGTTTTCGGTTGCGGTGACAAAAATTGGGCTACTACTTATCAAAAGGTTCCAGCCTTTATTGACGAAACTTTGGCTGCTAAAGGTGCTGAAAACATTGCTGACAGAGGTGAAGCTGATGCCTCCGACGACTTCGAAGGTACTTACGAAGAATGGAGAGAACACATGTGGTCTGACGTTGCTGCTTACTTCAACTTGGACATCGAAAACTCTGAAGACAACAAGTCCACTTTGTCTTTGCAATTCGTTGACTCCGCTGCTGACATGCCATTGGCTAAGATGCACGGTGCTTTCTCTACCAACGTCGTTGCCTCCAAGGAATTGCAACAACCAGGTTCTGCTAGATCTACTAGACACTTGGAAATCGAATTGCCAAAGGAAGCTTCCTACCAAGAAGGTGACCACTTGGGCGTTATTCCAAGAAACTACGAAGGTATCGTCAACAGAGTTACTGCTAGATTCGGTTTGGATGCTTCTCAACAAATCAGATTAGAAGCTGAAGAAGAAAAGTTGGCTCACTTGCCATTAGCTAAGACTGTCTCCGTTGAAGAATTGTTGCAATACGTCGAATTGCAAGACCCAGTTACCAGAACCCAATTGAGAGCCATGGCTGCCAAGACCGTCTGTCCACCACACAAGGTTGAATTGGAAGCCTTGTTGGAAAAGCAAGCCTACAAGGAACAAGTTTTGGCTAAGAGATTGACCATGTTGGAATTGTTGGAAAAGTACCCAGCCTGCGAAATGAAGTTCTCTGAATTTATCGCCTTGTTGCCATCTATCAGACCACGTTACTACTCTATTTCTTCCTCTCCACGTGTTGACGAAAAGCAAGCTTCTATTACTGTTTCCGTTGTCTCCGGTGAAGCTTGGTCCGGTTACGGTGAATACAAGGGTATTGCTTCTAACTACTTGGCTGAATTGCAAGAAGGTGACACCATTACTTGTTTCATCTCTACTCCACAATCCGAATTTACTTTGCCAAAGGACCCAGAAACTCCATTGATCATGGTTGGTCCAGGTACTGGTGTCGCTCCATTCAGAGGTTTCGTTCAAGCTAGAAAACAATTGAAGGAACAAGGTCAATCTTTGGGTGAAGCTCACTTGTACTTCGGTTGTAGATCTCCACACGAAGACTACTTATACCAAGAAGAATTGGAAAACGCTCAATCCGAAGGTATTATCACTTTGCACACCGCTTTCTCCAGAATGCCAAACCAACCAAAGACTTACGTCCAACACGTTATGGAACAAGACGGTAAGAAGTTGATTGAATTGTTGGACCAAGGTGCTCACTTCTACATTTGTGGTGATGGTTCTCAAATGGCTCCAGCCGTTGAAGCCACTTTGATGAAGTCTTACGCTGATGTTCACCAAGTTTCCGAAGCCGATGCTAGATTATGGTTGCAACAATTGGAAGAAAAAGGTCGTTAC GCTAAGGATGTCTGGGCCGGTTGA

TABLE 7 pA24, pA25, and pA26 sequences pA24cctcgccgcagttaattaaagtcagtgagcgaggaagcgcgtaactataacggtc SEQ ID Sequencectaaggtagcgaatcctgatgcggtattttctccttacgcatctgtgcggtattt NO: 112cacaccgcatagatcggcaagtgcacaaacaatacttaaataaatactactcagtaataacctatttcttagcatttttgacgaaatttgctattttgttagagtcttttacaccatttgtctccacacctccgcttacatcaacaccaataacgccatttaatctaagcgcatcaccaacattttctggcgtcagtccaccagctaacataaaatgtaagctttcggggctctcttgccttccaacccagtcagaaatcgagttccaatccaaaagttcacctgtcccacctgcttctgaatcaaacaagggaataaacgaatgaggtttctgtgaagctgcactgagtagtatgttgcagtcttttggaaatacgagtcttttaataactggcaaaccgaggaactcttggtattcttgccacgactcatctccatgcagtggagccaatcaattcttgcggtcaactttggacgatatcaatgccgtaatcattgaccagagccaaaacatcctccttaagttgattacgaaacacgccaaccaagtatttcggagtgcctgaactatttttatatgcttttacaagacttgaaattttccttgcaataaccgggtcaattgttctctttctattgggcacacatataatacccagcaagtcagcatcggaatctagagcacattctgcggcctctgtgctctgcaagccgcaaactttcaccaatggaccagaactacctgtgaaattaataacagacatactccaagctgcctttgtgtgcttaatcacgtatactcacgtgctcaatagtcaccaatgccctccctcttggccctctccttttcttttttcgaccgaattaattcttaatcggcaaaaaaagaaaagctccggatcaagattgtacgtaaggtgacaagctatttttcaataaagaatatcttccactactgccatctggcgtcataactgcaaagtacacatatattacgatgctgttctattaaatgcttcctatattatatatatagtaatgtcgtgatctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacggatcgcttgcctgtaacttacacgcgcctcgtatcttttaatgatggaataatttgggaatttactctgtgtttatttatttttatgttttgtatttggattttagaaagtaaataaagaaggtagaagagttacggaatgaagaaaaaaaaataaacaaaggtttaaaaaatttcaacaaaaagcgtactttacatatatatttattagacaagaaaagcagattaaatagatatacattcgattaacgataagtaaaatgtaaaatcacaggattttcgtgtgtggtcttctacacagacaaggtgaaacaattcggcattaatacctgagagcaggaagagcaagataaaaggtagtatttgttggcgatccccctagagtcttttacatcttcggaaaacaaaaactattttttctttaatttctttttttactttctatttttaatttatatatttatattaaaaaatttaaattataattatttttatagcacgtgatgaaaaggacccaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggacgcgtagtctagaccagccaggacagaaatgcctcgacttcgctgctacccaaggttgccgggtgacgcacaccgtggaaacggatgaaggcacgaacccagtggacataagcctgttcggttcgtaagctgtaatgcaagtagcgtatgcgctcacgcaactggtccagaaccttgaccgaacgcagcggtggtaacggcgcagtggcggttttcatggcttgttatgactgtttttttggggtacagtctatgcctcgggcatccaagcagcaagcgcgttacgccgtgggtcgatgtttgatgttatggagcagcaacgatgttacgcagcagggcagtcgccctaaaacaaagttaaacattatgagggaagcggtgatcgccgaagtatcgactcaactatcagaggtagttggcgccatcgagcgccatctcgaaccgacgttgctggccgtacatttgtacggctccgcagtggatggcggcctgaagccacacagtgatattgatttgctggttacggtgaccgtaaggcttgatgaaacaacgcggcgagctttgatcaacgaccttttggaaacttcggcttcccctggagagagcgagattctccgcgctgtagaagtcaccattgttgtgcacgacgacatcattccgtggcgttatccagctaagcgcgaactgcaatttggagaatggcagcgcaatgacattcttgcaggtatcttcgagccagccacgatcgacattgatctggctatcttgctgacaaaagcaagagaacatagcgttgccttggtaggtccagcggcggaggaactctttgatccggttcctgaacaggatctatttgaggcgctaaatgaaaccttaacgctatggaactcgccgcccgactgggctggcgatgagcgaaatgtagtgcttacgttgtcccgcatttggtacagcgcagtaaccggcaaaatcgcgccgaaggatgtcgctgccggctgggcaatggagcgcctgccggcccagtatcagcccgtcatacttgaagctagacaggcttatcttggacaagaagaagatcgcttggcctcgcgcgcagatcagttggaagaatttgtccactacgtgaaaggcgagatcaccaaggtagtcggcaaataaccctcgagcattcaaggcgccttgattatttgacgtggtttgatggcctccacgcacgttgtgatatgtagatgattcagttcgagtttatcattatcaatactgccatttcaaagaatacgtaaataattaatagtagtgattttcctaactttatttagtcaaaaaattagccttttaattctgctgtaacccgtacatgcccaaaatagggggcgggttacacagaatatataacatcgtaggtgtctgggtgaacagtttattcctgaaatattgttttcttcaccaaccatcagttcataggtccattctcttagcgcaactacagagaacaggggcacaaacaggcaaaaaacgggcacaacctcaatggagtgatgcaacctgcctggagtaaatgatgacacaaggcaattgacccacgcatgtatctatctcattttcttacaccttctattaccttctgctctctctgatttggaaaaagctgaaaaaaaaggttgaaaccagttccctgaaattattcccctacttgactaataagtatataaagacggtaggtattgattgtaattctgtaaatctatttcttaaacttcttaaattctacttttatagttagtcttttttttagttttaaaacaccaagaacttagtttcgaataaacacacataaacaaacaaaacaggccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctcctcccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttattttttttaatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacaaacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgtaatcattatcactttacgggtcctttccggtgatccgacaggttacggggcggcgacctcgcgggttttcgctatttatgaaaattttccggtttaaggcgtttccgttcttcttcgtcataacttaatgtttttatttaaaatacctcgcgagtggcaacactgaaaatacccatggagcggcgtaaccgtcgcacaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcagtggaacgtgcattatgaattagttacgctagggataacagggtaatatagaacccgaacgaccgagcgcagcggcggccgcgctga taccgccgc pA25aacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacg SEQ ID sequencecttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacag NO: 113gagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcagtggaacgtgcattatgaattagttacgctagggataacagggtaatatagaacccgaacgaccgagcgcagcggcggccgcgctgataccgccgccctcgccgcagttaattaaagtcagtgagcgaggaagcgcgtaactataacggtcctaaggtagcgaatcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatagatcggcaagtgcacaaacaatacttaaataaatactactcagtaataacctatttcttagcatttttgacgaaatttgctattttgttagagtcttttacaccatttgtctccacacctccgcttacatcaacaccaataacgccatttaatctaagcgcatcaccaacattttctggcgtcagtccaccagctaacataaaatgtaagctttcggggctctcttgccttccaacccagtcagaaatcgagttccaatccaaaagttcacctgtcccacctgcttctgaatcaaacaagggaataaacgaatgaggtttctgtgaagctgcactgagtagtatgttgcagtcttttggaaatacgagtcttttaataactggcaaaccgaggaactcttggtattcttgccacgactcatctccatgcagtggagccaatcaattcttgcggtcaactttggacgatatcaatgccgtaatcattgaccagagccaaaacatcctccttaagttgattacgaaacacgccaaccaagtatttcggagtgcctgaactatttttatatgcttttacaagacttgaaattttccttgcaataaccgggtcaattgttctctttctattgggcacacatataatacccagcaagtcagcatcggaatctagagcacattctgcggcctctgtgctctgcaagccgcaaactttcaccaatggaccagaactacctgtgaaattaataacagacatactccaagctgcctttgtgtgcttaatcacgtatactcacgtgctcaatagtcaccaatgccctccctcttggccctctccttttcttttttcgaccgaattaattcttaatcggcaaaaaaagaaaagctccggatcaagattgtacgtaaggtgacaagctatttttcaataaagaatatcttccactactgccatctggcgtcataactgcaaagtacacatatattacgatgctgttctattaaatgcttcctatattatatatatagtaatgtcgtgatctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacggatcgcttgcctgtaacttacacgcgcctcgtatcttttaatgatggaataatttgggaatttactctgtgtttatttatttttatgttttgtatttggattttagaaagtaaataaagaaggtagaagagttacggaatgaagaaaaaaaaataaacaaaggtttaaaaaatttcaacaaaaagcgtactttacatatatatttattagacaagaaaagcagattaaatagatatacattcgattaacgataagtaaaatgtaaaatcacaggattttcgtgtgtggtcttctacacagacaaggtgaaacaattcggcattaatacctgagagcaggaagagcaagataaaaggtagtatttgttggcgatccccctagagtcttttacatcttcggaaaacaaaaactattttttctttaatttctttttttactttctatttttaatttatatatttatattaaaaaatttaaattataattatttttatagcacgtgatgaaaaggacccaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggacgcgtagtctagaccagccaggacagaaatgcctcgacttcgctgctacccaaggttgccgggtgacgcacaccgtggaaacggatgaaggcacgaacccagtggacataagcctgttcggttcgtaagctgtaatgcaagtagcgtatgcgctcacgcaactggtccagaaccttgaccgaacgcagcggtggtaacggcgcagtggcggttttcatggcttgttatgactgtttttttggggtacagtctatgcctcgggcatccaagcagcaagcgcgttacgccgtgggtcgatgtttgatgttatggagcagcaacgatgttacgcagcagggcagtcgccctaaaacaaagttaaacattatgagggaagcggtgatcgccgaagtatcgactcaactatcagaggtagttggcgccatcgagcgccatctcgaaccgacgttgctggccgtacatttgtacggctccgcagtggatggcggcctgaagccacacagtgatattgatttgctggttacggtgaccgtaaggcttgatgaaacaacgcggcgagctttgatcaacgaccttttggaaacttcggcttcccctggagagagcgagattctccgcgctgtagaagtcaccattgttgtgcacgacgacatcattccgtggcgttatccagctaagcgcgaactgcaatttggagaatggcagcgcaatgacattcttgcaggtatcttcgagccagccacgatcgacattgatctggctatcttgctgacaaaagcaagagaacatagcgttgccttggtaggtccagcggcggaggaactctttgatccggttcctgaacaggatctatttgaggcgctaaatgaaaccttaacgctatggaactcgccgcccgactgggctggcgatgagcgaaatgtagtgcttacgttgtcccgcatttggtacagcgcagtaaccggcaaaatcgcgccgaaggatgtcgctgccggctgggcaatggagcgcctgccggcccagtatcagcccgtcatacttgaagctagacaggcttatcttggacaagaagaagatcgcttggcctcgcgcgcagatcagttggaagaatttgtccactacgtgaaaggcgagatcaccaaggtagtcggcaaataaccctcgagcattcaaggcgccttgattatttgacgtggtttgatggcctccacgcacgttgtgatatgtagatgagagcgttggttggtggatcaagcccacgcgtaggcaatcctcgagcagatccgccaggcgtgtatatatagcgtggatggccaggcaactttagtgctgacacatacaggcatatatatatgtgtgcgacaacacatgatcatatggcatgcatgtgctctgtatgtatataaaactcttgttttcttcttttctctaaatattctttccttatacattaggacctttgcagcataaattactatacttctatagacacacaaacacaaatacacacactaaattaataacaggccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacaaacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgtaatcattatcactttacgggtcctttccggtgatccgacaggttacggggcggcgacctcgcgggttttcgctatttatgaaaattttccggtttaaggcgtttccgttcttcttcgtcataacttaatgtttttatttaaaatacctcgcgagtggcaacactgaaaatacccatggagcggcgtaaccgtcgcacaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcg pA26acgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgc SEQ ID sequencettcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagg NO: 114agagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcagtggaacgtgcattatgaattagttacgctagggataacagggtaatatagaacccgaacgaccgagcgcagcggcggccgcgctgataccgccgccctcgccgcagttaattaaagtcagtgagcgaggaagcgcgtaactataacggtcctaaggtagcgaatcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatagatcggcaagtgcacaaacaatacttaaataaatactactcagtaataacctatttcttagcatttttgacgaaatttgctattttgttagagtcttttacaccatttgtctccacacctccgcttacatcaacaccaataacgccatttaatctaagcgcatcaccaacattttctggcgtcagtccaccagctaacataaaatgtaagctttcggggctctcttgccttccaacccagtcagaaatcgagttccaatccaaaagttcacctgtcccacctgcttctgaatcaaacaagggaataaacgaatgaggtttctgtgaagctgcactgagtagtatgttgcagtcttttggaaatacgagtcttttaataactggcaaaccgaggaactcttggtattcttgccacgactcatctccatgcagtggagccaatcaattcttgcggtcaactttggacgatatcaatgccgtaatcattgaccagagccaaaacatcctccttaagttgattacgaaacacgccaaccaagtatttcggagtgcctgaactatttttatatgcttttacaagacttgaaattttccttgcaataaccgggtcaattgttctctttctattgggcacacatataatacccagcaagtcagcatcggaatctagagcacattctgcggcctctgtgctctgcaagccgcaaactttcaccaatggaccagaactacctgtgaaattaataacagacatactccaagctgcctttgtgtgcttaatcacgtatactcacgtgctcaatagtcaccaatgccctccctcttggccctctccttttcttttttcgaccgaattaattcttaatcggcaaaaaaagaaaagctccggatcaagattgtacgtaaggtgacaagctatttttcaataaagaatatcttccactactgccatctggcgtcataactgcaaagtacacatatattacgatgctgttctattaaatgcttcctatattatatatatagtaatgtcgtgatctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacggatcgcttgcctgtaacttacacgcgcctcgtatcttttaatgatggaataatttgggaatttactctgtgtttatttatttttatgttttgtatttggattttagaaagtaaataaagaaggtagaagagttacggaatgaagaaaaaaaaataaacaaaggtttaaaaaatttcaacaaaaagcgtactttacatatatatttattagacaagaaaagcagattaaatagatatacattcgattaacgataagtaaaatgtaaaatcacaggattttcgtgtgtggtcttctacacagacaaggtgaaacaattcggcattaatacctgagagcaggaagagcaagataaaaggtagtatttgttggcgatccccctagagtcttttacatcttcggaaaacaaaaactattttttctttaatttcggacccaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggacgcgtagtctagaccagccaggacagaaatgcctcgacttcgctgctacccaaggttgccgggtgacgcacaccgtggaaacggatgaaggcacgaacccagtggacataagcctgttcggttcgtaagctgtaatgcaagtagcgtatgcgctcacgcaactggtccagaaccttgaccgaacgcagcggtggtaacggcgcagtggcggttttcatggcttgttatgactgtttttttggggtacagtctatgcctcgggcatccaagcagcaagcgcgttacgccgtgggtcgatgtttgatgttatggagcagcaacgatgttacgcagcagggcagtcgccctaaaacaaagttaaacattatgagggaagcggtgatcgccgaagtatcgactcaactatcagaggtagttggcgccatcgagcgccatctcgaaccgacgttgctggccgtacatttgtacggctccgcagtggatggcggcctgaagccacacagtgatattgatttgctggttacggtgaccgtaaggcttgatgaaacaacgcggcgagctttgatcaacgaccttttggaaacttcggcttcccctggagagagcgagattctccgcgctgtagaagtcaccattgttgtgcacgacgacatcattccgtggcgttatccagctaagcgcgaactgcaatttggagaatggcagcgcaatgacattcttgcaggtatcttcgagccagccacgatcgacattgatctggctatcttgctgacaaaagcaagagaacatagcgttgccttggtaggtccagcggcggaggaactctttgatccggttcctgaacaggatctatttgaggcgctaaatgaaaccttaacgctatggaactcgccgcccgactgggctggcgatgagcgaaatgtagtgcttacgttgtcccgcatttggtacagcgcagtaaccggcaaaatcgcgccgaaggatgtcgctgccggctgggcaatggagcgcctgccggcccagtatcagcccgtcatacttgaagctagacaggcttatcttggacaagaagaagatcgcttggcctcgcgcgcagatcagttggaagaatttgtccactacgtgaaaggcgagatcaccaaggtagtcggcaaataaccctcgagcattcaaggcgccttgattatttgacgtggtttgatggcctccacgcacgttgtgatatgtagatgactcgtaggaacaatttcgggcccctgcgtgttcttctgaggttcatcttttacatttgcttctgctggataattttcagaggcaacaaggaaaaattagatggcaaaaagtcgtctttcaaggaaaaatccccaccatctttcgagatcccctgtaacttattggcaactgaaagaatgaaaaggaggaaaatacaaaatatactagaactgaaaaaaaaaaagtataaatagagacgatatatgccaatacttcacaatgttcgaatctattcttcatttgcagctattgtaaaataataaaacatcaagaacaaacaagctcaacttgtcttttctaagaacaaagaataaacacaaaaacaaaaagtttttttaattttaatcaaaaaacaggccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacaaacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgtaatcattatcactttacgggtcctttccggtgatccgacaggttacggggcggcgacctcgcgggttttcgctatttatgaaaattttccggtttaaggcgtttccgttcttcttcgtcataacttaatgtttttatttaaaatacctcgcgagtggcaacactgaaaatacccatggagcggcgtaaccgtcgcacaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcga

TABLE 8 Tailoring enzymes Reaction Catalyzed Enzyme SpeciesCarbon-carbon Berberine bridge enzyme (BBE) Ps, Ec, Cj, Bs, Tf couplingSaluteridine synthase (SalSyn) Ps Corytuberine synthase (CorSyn) CjOxidation Tetrahydroprotoberberberine Cj, Am, Bw oxidase (STOX)Dihydrobenzophenanthridine Ps oxidase (DBOX) Methylstylopine hydroxylasePs (MSH) Protopine 6-hydroxylase (P6H) Ps, Ec Methylenedioxy Stylopinesynthase (StySyn) Ps, Ec, Am bridge Chellanthifoline synthase Ps, Ec, Amformation (CheSyn) Canadine synthase (CAS) Tf, Cc O-methylationNorcoclaurine Ps, Tf, Cj, Pb 6-O-methyltransferase (6OMT) 3′hydroxy-N-Ps, Tf, Cj, Cc methylcoclaurine 4′-O- methyltransferase (4′OMT)Reticuline 7-O-methyltransferase Ps, Ec (7OMT) Scoulerine9-O-methyltransferase Ps, Tf, Cj, Cc (9OMT) N-methylation CoclaurineN-methyltransferase Ps, Tf, Cj (CNMT) Tetrahydroprotoberberine N- Ps,Ec, Pb methyltransferase (TMNT) O-demethylation Thebaine demethylase(T6ODM) Ps Codeine demthylase (CODM) Ps, Ga Reduction Salutaridinereductase (SalR) Ps, Pb, Ga Codeinone reductase (COR) Ps Sanguinarinereductase (SanR) Ec Acetylation Salutaridine acetyltransferase Ps(SalAT)

Comparison of impurities that may be present in concentrate of poppystraw and clarified yeast culture medium. Con- Clarified centrate Yeastof Poppy Culture Impurities Straw Medium Inorganic Sodium ✓ ✓ Magnesium✓ ✓ Silicon ✓ ϰ ( 

 ) Phosphorus ✓ ✓ Sulfur ✓ ✓ Chloride ✓ ✓ Potassium ✓ ✓ Calcium ✓ ✓Copper ✓ ✓ Zinc ✓ ✓ Molybdenum ✓ ✓( 

 ) Iron ✓ ✓ Manganese ✓ ✓ Ammonium ✓ ✓ Boron ✓ ✓ Organic Polysaccharides✓ ϰ (

) (starch, cellulose, xylan) Lignin (p-coumaryl, ✓ ϰ coniferyl, sinapylalcohols) Pigments (chlorophyll, ✓ ϰ anthocyanins, carotenoids)Flavonoids ✓ ϰ Phenanthreoids ✓ ϰ Latex, gum, and wax ✓ ϰ Rubisco ✓ ϰMeconic acid ✓ ϰ Pseudomorphine ✓ ϰ Narceine ✓ ϰ Thebanol ✓ ϰ OtherPesticides ✓ ϰ Pollen ✓ ϰ

indicates data missing or illegible when filed

TABLE 10 Distinct groups of molecules present in clarified yeast culturemedium (CYCM). Unlike concentrate of poppy straw (CPS), yeast hoststrains may be engineered to produce molecules of a predetermined classof alkaloids (i.e., only one biosynthesis pathway per strain) such thatother classes of alkaloids are not present. Therefore, the CYCM maycontain molecules within a single biosynthesis pathway including asubset of molecules spanning one or two columns, whereas the CPS maycontain a subset of molecules across many columns. Protoberberine and1-Benzylisoquinoline Phthalidesoquinoline Morphinan Isopavine AporphineBisBIA Tetrahydropapaverine Scoulenner

Pavine

Dauricine Dihydropapeverine

Caryachine

Papaverine Stylopine

Cis-N-methylstylopine Thebaine

Boidine Fangchinoline Protopine

Tetrandine Sanguinarine Ospavine Curinet Tetrahydrocolumbine MorphinoneCepharanthine Canadine Neopinone Bernemine N-methylcanadine CodeineNoscapine Morphine Berberine Neomorphine Hydrocodine Oxycodone14-hydroxycodenone 14-hydroxycodeine Dihydromorphine Dihydrocodeine

indicates data missing or illegible when filed

TABLE 11 Impurities that may be present in chemical synthesispreparations of compounds Compound Impurities Buprenorphine15,16-Dehydrobuprenorphine, 17,18- Dehydrobuprenorphine,18,19-demethylbuprenorphine, 19,19′-Ethylbuprenorphine,2,2′-Bisbuprenorphine, 3- Deshydroxybuprenorphine,3-O-Methylbuprenorphine, 3-O- Methyl-N-cyanonorbuprenorphine,3-O-Methyl-N- methylnorbuprenorphine, 6-O-Desmethylbuprenorphine,Buprenorphine N-oxide, N-But-3-enylnorbuprenorphine, N-But-3-enylnormethylbuprenorphine, N- Butylnorbuprenorphine,N-Methylbuprenorphine, Norbuprenorphine, Tetramethylfuran buprenorphineOxymorphone 1-Bromooxymorphone, 6-Beta oxymorphol, 10-Alpha-hydroxyoxymorphone, 10-Ketooxymorphone, 2,2- Bisoxymorphone,Noroxymorphone, Oxymorphone N-oxide, 10-Hydroxyoxymorphone,4-Hydroxyoxymorphone, 8- Hydroxyoxymorphone, Hydromorphinol. Naltrexone10-Hydroxynaltrexone, 10-Ketonaltrexone, 14-Hydroxy-17-cyclopropylmethylnormorphinone, 2,2′-Bisnaltrexone, 3-Cyclopropylmethylnaltrexone, 3-O-Methylnaltrexone, 8- Hydroxynaltrexone,N-(3-Buteny1)-noroxymorphone, Naltrexone aldol dimer,N-Formyl-noroxymorphone Naloxone 10-Alpha-hydroxynaloxone,10-Beta-hydroxynaloxone, 10- Ketonaloxone, 3-O-Allylnaloxone,7,8-Didehydronaloxone, 2,2′-Bisnaloxone, Naloxone N-oxide NalbuphineBeta-epimer of nalbuphine, 2,2′-Bisnalbuphine, 6-Ketonalbuphine,10-Ketonalbuphine, Alpha-noroxymorphol, N-(Cyclobutylcarbony1)-alpha-noroxymorphol, N-Formy1-6-alpha-noroxymophol.

While preferred embodiments of the invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A method of demethylating a first opioid to a second opioid,comprising: contacting the first opioid with at least one enzyme,wherein contacting the first opioid with the at least one enzymeconverts the first opioid to the second opioid through loss of anO-linked methyl group, and wherein the first opioid is not selected fromthe group consisting of codeine and thebaine.
 2. A method ofdemethylating an opioid to a nor-opioid, comprising: contacting a firstopioid with at least one enzyme, wherein contacting the first opioidwith the at least one enzyme converts the first opioid to a secondopioid through loss of an O-linked methyl group; and contacting thesecond opioid with at least one enzyme, wherein contacting the opioidwith the at least one enzyme converts the second opioid to a nor-opioidthrough loss of an N-linked methyl group.
 3. A method of demethylatingan opioid to a nor-opioid, comprising: contacting the opioid with atleast one enzyme, wherein contacting the opioid with the at least oneenzyme converts the opioid to the nor-opioid through removal of aN-linked methyl group from the opioid, wherein the opioid is notthebaine when the opioid contacts the at least one enzyme in vitro. 4.(canceled)
 5. (canceled)
 6. The method of claim 1, wherein the secondopioid is produced by culturing an engineered cell comprising a codingsequence for encoding the at least one enzyme.
 7. The method of claim 2,wherein the nor-opioid is produced by culturing an engineered cellcomprising a coding sequence for encoding the first enzyme.
 8. Themethod of claim 3, wherein the nor-opioid is produced by culturing anengineered cell comprising a coding sequence for encoding the firstenzyme and the second enzyme.
 9. (canceled)
 10. (canceled)
 11. Themethod of claim 6, further comprising: recovering the second opioid fromthe cell culture.
 12. The method of claim 7, further comprising:recovering the nor-opioid from the cell culture.
 13. (canceled)
 14. Themethod of claim 6, further comprising: adding a (S)-1-benzylisoquinolinealkaloid to the cell culture.
 15. The method of claim 7, wherein thenor-opioid is produced within an engineered cell by a metabolic pathwaystarting with L-tyrosine. 16.-22. (canceled)
 23. The method of claim 6,wherein the engineered cell is an engineered non-plant cell.
 24. Themethod of claim 23, wherein the engineered non-plant cell is anengineered yeast cell.
 25. The method of claim 1, wherein the loss ofthe O-linked methyl group occurs at the 3′ position. 26.-40. (canceled)41. The method of claim 6, wherein said engineered cell comprises aheterologous coding sequence, wherein said heterologous coding sequenceencodes an enzyme selected from the group consisting of N-demethylases.42.-67. (canceled)
 68. The method of claim 8, further comprising:recovering the nor-opioid from the cell culture.
 69. The method of claim8, wherein the nor-opioid is produced within an engineered cell by ametabolic pathway starting with L-tyrosine.
 70. The method of claim 7,wherein the engineered cell is an engineered non-plant cell.
 71. Themethod of claim 70, wherein the engineered non-plant cell is anengineered yeast cell.
 72. The method of claim 2, wherein the loss ofthe O-linked methyl group occurs at the 3′ position.
 73. The method ofclaim 8, wherein the nor-opioid is produced within an engineered cell bya metabolic pathway starting with L-tyrosine.